Methods for reducing intraocular pressure

ABSTRACT

A method for decreasing intraocular pressure by administering an A 3  subtype adenosine receptor antagonist, a calmodulin antagonist or an antiestrogen such as tamoxifen. These agents, by inhibiting influx or promoting efflux of aqueous humor, can be used to treat glaucoma.

FIELD OF THE INVENTION

[0001] The present invention relates to the use of A3 subtype adenosinereceptor antagonists, calmodulin antagonists and antiestrogens forreduction of intraocular pressure.

BACKGROUND OF THE INVENTION

[0002] The aqueous humor of the eye is formed by the ciliary epithelium,which comprises two cell layers: the outer pigmented epithelial (PE)cells facing the stroma and the inner nonpigmented epithelial (NPE)cells in contact with the aqueous humor. Secretion is generally thoughtto reflect a primary transfer of solute, largely NaCl, from the stromato the aqueous humour, providing an osmotic driving force for thesecondary osmotic transfer of water down its chemical gradient (Cole,.Exp. Eye Res. 25 (Suppl.), 161-176, 1977), although a more directcoupling between water and solute may also proceed across epithelia(Meinild et al. J. Physiol. 508: 15-21, 1998). One major factorgoverning the rate of secretion is the rate of chloride ion (Cl⁻)releasefrom the NPE cells into the aqueous humor (Civan, News Physiol. Sci.12:158-162, 1997). The activity of Cl⁻ channels is likely to be arate-limiting factor in aqueous humour secretion, given the low baselinelevel of channel activity and the predominance of the chloride anion inthe fluid transferred (Coca-Prados et al., Am. J. Physiol. 268:C572-C579, 1995). The secretion of aqueous humor into the eye isbelieved to result as a consequence of two opposing physiologicalprocesses: fluid secretion into the eye by the NPE cells and fluidreabsorption (secretion out of the eye) by the PE cells. Thus, bothrelease of Cl⁻ by the nonpigmented ciliary epithelial (NPE) cells intothe adjacent aqueous humour would enhance secretion, and Cl⁻ release bythe pigmented ciliary epithelial (PE) cells into the neighboring stromawould reduce net secretion (Civan, Current Topics in Membranes 45: 1-24,1998).

[0003] Recently, adenosine has been found to activate NPE Cl⁻ channelswhich subserve this release (Carre et al., Am. J. Physiol. (CellPhysiol. 42) 273:C1354-C1361, 1997). Purines, a class of chemicalcompounds which includes adenosine, ATP and related compounds, mayregulate aqueous humour secretion, in part through modifying Cl⁻-channelactivity. Both NPE and PE cells have been reported to release ATP to theextracellular surface, where ATP can be metabolized to adenosine byecto-enzymes (Mitchell et al. Proc. Natl. Acad. Sci U.S.A. 95:7174-7178, 1998), and both cell types possess adenosine receptors (Waxet al., Exp. Eye Res. 57:89-95, 1993; Wax et al, Invest. Ophthalmol.Vis. Sci., 35:3057-3063, 1994; Kvanta et al., Exp. Eye Res. 65:595-602,1997) and ATP receptors (Wax et al., supra., 1993; Shahidullah et al.,Curr. Eye Res. 16:1006-1016, 1997). Furthermore, in vitro studies ofrabbits have associated A₂-adenosine receptors with increased secretionand elevated intraocular pressure (Crosson et al., Invest. Ophthalmol.Vis. Sci. 37:1833-1839, 1996) and A₁-adenosine receptors with theconverse (Crosson, J. Pharmacol. Exp. Ther. 273:320-326, 1995).Qualitatively similar associations with intraocular pressure, but notwith secretion, have been observed in cynomologus monkeys (Tain et al.,Exp. Eye Res., 64: 979-989, 1997). A particular role for Cl⁻ channelshas been suggested by the observations that adenosine agonists stimulateCl⁻ channels of immortalized human and freshly-dissected bovine NPEcells and of aqueous-oriented Cl⁻ channels of the intact rabbitiris-ciliary body (Carré et al., supra., 1997). Adenosine triggeredisotonic shrinkage of cultured human cells from the HCE cell line. Thecontribution of Cl⁻ channels to this shrinkage was identified byperforming the experiments in the presence of the cation ionophoregramicidin. In addition, adenosine produced a Cl⁻ dependent increase inshort-circuit current across rabbit iris-ciliary body while thenon-metabolizable adenosine analogue 2-Cl-adenosine was shown toactivate Cl⁻ currents in HCE cells using the whole cell patch-clamptechnique. Although this study clearly established that adenosine couldactivate Cl⁻ channels on NPE cells, the concentrations of agonist usedwere capable of stimulating all four known adenosine receptor sub-types:A₁, A_(2A), A_(2B) and A₃ (Fredholm et al., Pharmacol. Rev., 46:143-156,1994; Fredholm et al., Trends Pharmacol. Sci, 18:79-82, 1997; Klotz etal., Naunyn Schmiedebergs Arch. Pharmacol., 357:1-9., 1998). Ciliaryepithelial cells are known to possess A₁ A_(2A) and A_(2B) adenosinereceptors (Kohno et al., Blood, 88:3569-3574, 1996, Stambaugh et al.,Am. J. Physiol. 273 (Heart Circ. Physiol. 42):H501-H505, 1997; Wax etal., supra., 1994). Although stimulation of these receptors can beassociated with specific changes in the levels of second messengers cAMP(Crosson, supra.; Stambaugh et al., supra.; Wax et al., supra., 1994)and Ca²⁺ (Farahbakhsh et al., Exp. Eye Res., 64:173-179, 1997), theeffect of these receptors upon Cl⁻ channels of NPE cells was unknown.

[0004] Alternatively, the intraocular pressure could be reduced bystimulating reabsorption of aqueous humor. In principle, this could beachieved by activating chloride channels on the basolateral surface ofthe pigmented cell layer. This would release chloride back into thestroma. One way to accomplish this with the PE cells has been identifiedusing the antiestrogen tamoxifen. Tamoxifen is known to exert multipleactions on biological cells. However, recently, Mitchell et al. (InvestOphthalmol. Vis. Sci. 38(Suppl.):S1042, 1997) have noted that the onlyknown action of tamoxifen which could account for the phenomenon is itsantiestrogenic activity, probably on the plasma membrane.

[0005] Glaucoma is a disorder characterized by increased intraocularpressure that may cause impaired vision, ranging from slight loss toabsolute blindness. The increased intraocular pressure is related to animbalance between production and outflow of the aqueous humor. Currentdrugs prescribed for glaucoma, in the form of eyedrops, includepilocarpine, timolol, betaxolol, levobunolol, metipranolol, epinephrine,dipivefrin, latanoprost, carbachol, and potent cholinesterase inhibitorssuch as echothiophate and carbonic anhydrase inhibitors such asdorzolamidet. Many of these effective approaches to medical therapy ofglaucoma involve a reduction in the rate of flow into the eye. However,none of these drugs are satisfactory, in part due to side effects.

[0006] Because of side effects of available agents and inconvenientdosing schedule, there is an ongoing need for compounds capable ofreducing intraocular pressure for the treatment of glaucoma withimproved efficacy, prolonged action and reduced side effects. Thepresent invention addresses this need.

SUMMARY OF THE INVENTION

[0007] One embodiment of the present invention is a method for reducingintraocular pressure in an individual, comprising the step ofadministering to the individual an effective intraocularpressure-reducing amount of a pharmaceutical composition comprising anA₃ subtype adenosine receptor antagonist. In one aspect of thispreferred embodiment, the A₃ receptor antagonist is a dihydropyridine,pyridine, pyridinium salt or triazoloquinazoline. Preferably, the A₃subtype receptor antagonist is selected from the group consisting ofMRS-1097, MRS-1191, MRS-1220 and MRS-1523. Advantageously, thepharmaceutical composition is administered topically, systemically ororally. Preferably, the pharmaceutical composition is an ointment, gelor eye drops.

[0008] Another embodiment of the present invention is a method forreducing intraocular pressure in an individual, comprising the step ofadministering to the individual an effective intraocularpressure-reducing amount of a pharmaceutical composition comprising anantiestrogen. Preferably, the antiestrogen is tamoxifen. Advantageously,the pharmaceutical composition is administered topically, systemicallyor orally. Preferably, the pharmaceutical composition is ointment, gelor eye drops.

[0009] The present invention also provides a method for reducingintraocular pressure in an individual, comprising the step ofadministering to the individual an effective intraocularpressure-reducing amount of a pharmaceutical composition comprising acalmodulin antagonist. Preferably, the calmodulin antagonist istrifluoperazine. Advantageously, the pharmaceutical composition isadministered topically, systemically or orally. Preferably, thepharmaceutical composition is ointment, gel or eye drops.

[0010] Another embodiment of the present invention is an A₃ subtypeadenosine receptor antagonist for use in reduction of intraocularpressure. Preferably, the A₃ subtype adenosine receptor antagonist isMRS-1097, MRS-1191 or MRS-1523.

[0011] The present invention also provides the use of an antiestrogen inthe preparation of a medicament for the reduction of intraocularpressure. Preferably, the antiestrogen is tamoxifen.

[0012] The present invention also provides a calmodulin antagonist foruse in reduction of intraocular pressure. Preferably, the calmodulinantagonist is trifluoperazine

BRIEF DESCRIPTION OF THE FIGURES

[0013]FIG. 1 is a schematic diagram showing the ocular nonpigmentedepithelial (NPE) and pigmented epithelial (PE) cells, and the effects ofATP, adenosine (Ado) and tamoxifen (TMX) on the movement of aqueoushumor. Ecto=ecto-enzymes; A₃=A₃ subtype adenosine receptor.

[0014] FIGS. 2-7 relate to the effects of A₃ receptors on NPE cells,while FIGS. 8-17 relate primarily to the actions of ATP on PE cells.

[0015] FIGS. 2A-D show the concentration-response relationship for theA₃-selective agonist N⁶-(3-iodobenzyl)-adenosine-5′-N-methyluronamide(IB-MECA)-stimulated isotonic shrinkage of NPE cells in the presence of5 μM gramicidin. In FIGS. 2-5, solid trajectories are least-square fitswith monoexponentials, whereas data sets displaying no significantshrinkage are connected by dotted lines.

[0016]FIG. 2A:—least-squares fits yielded the following estimated valuesfor data obtained in parallel at concentrations of 30 nM-1 υM IB-MECA(n=4 experiments): 30 nM [steady-state cell volume (V∞)=98.5±0.1%,τ=3.3±1.5 min), and 1 μM (V∞=95.9±0.3%, τ=2.4±0.7 min). Control and 3sets of experimental results were significantly different (P<0.01,F-test).

[0017]FIG. 2B—data obtained over concentration range of 1-10 μM IB-MECA(n=4): 1 μM (V∞=97.5±0.1%, τ=7.2±1.0 min), 3 μM (V∞=96.7±0.3%,τ=10.0±2.3 min), and 10 μM (V∞=97.9±0.2%, τ=3.2±1.1 min). Data obtainedat 1 μM and 3 μM did not significantly deviate from the fit obtainedwith 10 μM IB-MECA.

[0018]FIG. 2C shows a Lineweaver-Burk plot generated from nonlinearleast-square fits of FIGS. 1A and 1B. Change in volume was calculated as(VO-V∞). Variation with passage number was noted for τ and (V0-V∞). Forthis reason, both experimental sets (FIGS. 1A and 1B) includedmeasurements with one concentration (1 μM) in common. Ratio (in V0-V∞)obtained at 1 μM in B to A was used as a scaling factor to accommodateresults obtained in B with 3 and 10 μM. Using this approach, linearleast-squares analysis (r²=0.95) led to an estimated value for theapparent K_(d) of 55±10 nM.

[0019]FIG. 2D shows isotonic cell shrinkage stimulated by 100 nMCl-IB-MECA. Fits for Cl-IB-MECA (V∞=97.9±0.2%, τ=2.5±1.3 min) andIB-MECA (V∞=96.8±0.4%, τ=6.3±2.2 min) are not significantly different(P>0.05).

[0020] FIGS. 3A-3B show the effect of A₃ antagonists on theIB-MECA-stimulated isotonic shrinkage of NPE cells. FIG. 3A shows thatthe A₃-selective antagonist MRS-1097 (300 nM) prevented shrinkagetriggered by IB-MECA (P<0.01, F-distribution). FIG. 3B shows that theA₃-selective antagonist MRS-1191 (100 nM) prevented characteristicshrinkage triggered by IB-MECA (n=4, P<0.01 by F-distribution). MRS didnot affect cell volume in the absence of IB-MECA, confirming thespecificity of the interaction (n=4).

[0021] FIGS. 4A-4C shows the effects of selective A₃-receptorantagonists on adenosine-stimulated isotonic shrinkage of NPE cells.Application of 300 nM MRS-1097 (FIG. 3A; n=4), 100 nM MRS-1191 (FIG. 3B;n=3), and 100 nM MRS-1523 (FIG. 3C; n=3) all prevented thecharacteristic shrinkage triggered by nonselective activation ofadenosine receptors with 10 μM adenosine (P<0.01, F-distribution).

[0022] FIGS. 5A-5C show the effects of adenosine-receptor agonists onisoosmotic volume of NPE cells. In FIG. 5A, the A₃-selective agonistIB-MECA produced prompt shrinkage at 100 nM (n=4, V∞=95.6±0.2%, τ4.5±0.6min, P<0.01 by F-distribution). In contrast, the A₁-selective agonistN⁶-cyclopentyladenosine (CPA) had little effect at 100 nM, and none atall at 3 μM (n=4). In FIG. 5B, at 100 nM, the A₂-selective agonistCGS-21680 exerted no effect, but the A₃-selective agonist IB-MECA againproduced shrinkage (n=4, P<0.01 by F-distribution). In FIG. 5C, at highconcentration (3 μM), the A₂-selective agonist CGS-21680 also triggeredisoosmotic shrinkage. However, preincubation of the cells with theselective A₃ receptor antagonist MRS-1191 (100 nM) abolished this effect(n=4, P<0.01, F-distribution).

[0023]FIG. 6 shows the effects of IB-MECA on the level of freeintracellular calcium of NPE cells. Concentration of intracellular Ca²⁺increased steadily after application of 100 nM IB-MECA and returned tobaseline levels once IB-MECA was removed. Data were obtained at asampling rate of 1 Hz and smoothed by 21 points. The box indicates theduration of the IB-MECA application.

[0024]FIG. 7 shows the effect of IB-MECA on short-circuit current(I_(SC)) across intact rabbit ciliary epithelium. As an initial step indata analysis, 20-min period of baseline current just before addition ofany agent was fit by linear least-squares analysis. The line generatedby that analysis was extrapolated to a point 45 min beyond introductionof that agent. Each current response was subtracted from its respectiveextrapolated baseline to yield a common initial baseline approximatingconstant zero current. All recordings were placed in register relativeto time of agent introduction (time 0). Records of control (solvent),IB-MECA with solvent, and B-MECA corrected for solvent were separatelyaveraged. IB-MECA was always added in the presence of 5 nM Ba²⁺ toisolate contribution of Cl⁻ to the response.

[0025] FIGS. 2-7 show that A₃-selective adenosine receptors increasechloride channel activity of NPE cells, and that blocking of thesereceptors reduces chloride channel activity and secretion by the NPEcells into the aqueous humor.

[0026] FIGS. 8-17 provide data to support an alternative approach toreducing net aqueous humor secretion and intraocular pressure byenhancing reabsorption by the PE cells.

[0027] FIGS. 8A-8C show the dependence of PE cell volume on ATPconcentration. In all figures, the volumes are normalized to the initialvalues. The nonlinear least square fits are presented as uninterruptedor interrupted curves, and data points displaying no significantshrinkage are connected by dotted lines.

[0028]FIG. 8A shows that in ˜15% of the preparations, aconcentration-dependent shrinkage was observed with ATP alone (N=4,P<0.01). The values generated by the fits were: v_(∞)=94.3±0.3% andτ=2.8±0.7 min (10 μM ATP), and v_(∞)=94.3±0.3%, τ=2.8±0.7 min (100 μMATP).

[0029]FIG. 8B shows the combined effect of tamoxifen (TMX) and ATP onsuspensions responding to ATP alone. The presence of 10 μM tamoxifenenhanced the response to both 3 μM (P<0.01) and 10 μM ATP (P<0.05). Thevalues of the fits were: v_(∞)=98.0±0.4%, τ=10.8±4.9 min (3 μM ATPalone); v_(∞)=96.7±0.4%, τ=5.8±2.1 min (3 μM ATP+10 μM TMX);v_(∞)=97.2±0.3%, τ=1.6±1.0 min (10 μM ATP alone); v_(∞)=95.7±0.3%,τ=1.4±0.7 min (10 μM ATP+10 μM TMX).

[0030]FIG. 8C shows the dependence of synergistic shrinkage on ATPconcentration. Tamoxifen (6 μM) was present throughout (N=5). Shrinkagewas observed in the simultaneous presence of 10 μM or 1 mM ATP (P<0.01),but not at 1 μM ATP. The data obtained at 10 μM and 1 mM ATP were notsignificantly different from each another. The fit obtained at 10 μM ATPwas characterized by v_(∞)=95.7±0.7% and τ=6.6±3.2 min.

[0031] FIGS. 9A-9C show the effects of ATP and NPPB on whole-cellcurrents of PE cells. The solid bars above show the time period of drugapplication. In FIG. 9A, at a holding potential of −60 mV, 1 mM ATPproduced reversible and reproducible increases in inward current in thecultured bovine PE cell of the panel. In FIG. 9B, the negatively-chargedchloride-channel blocker NPPB partially inhibited the ATP-stimulatedcurrent of the cell of the cell patched, even at −60 mV. In FIG. 9C, inthe freshly-dissociated cell of the panel, increasing concentrations ofATP (10 μM, 100 μM and 1 mM) elicited increasing large stimulations,partially reversible at 1 mM before losing the seal. These effects ofATP alone were observed only in half of the total cultured andfreshly-dissociated cells studied.

[0032] FIGS. 10A-10B. show the synergism between tamoxifen and ATP onisosmotic cell volume of PE cells. In FIG. 10A, neither ATP (10 mM) nortamoxifen (6 μM) separately produced substantial shrinkage, whereas even10 μM ATP added together with 6 μM tamoxifen substantially enhanced thebaseline shrinkage. The values of the fits were: v_(∞)=96.2±0.6%,τ=15.9±5.2 min (Control), 97.7±0.2%, τ=2.2±0.8 min (10 mM ATP alone),v_(∞)=97.2±0.3%, τ=1.6±1.0 min (10 μM ATP alone); and v_(∞)=92.8±0.8%,τ=11.4±0.3 min (10 μM ATP+6 μM TMX) (N=6, P<0.01). In contrast, in FIG.10B, no such synergism was noted between tamoxifen (6 μM) and adenosine(10 μM) (N=4).

[0033]FIG. 11 shows the effect of tamoxifen on the regulatory volumedecrease (RVD) of NPE cells. Gramicidin D (5 μM) was present in allsuspensions to provide an exit pathway for K⁺. In the absence oftamoxifen (TMX), the NPE cells displayed a regulatory volume response tohypotonic swelling with a half-time of ˜5 min (N=5). Adding TMX at theconclusion of the RVD (t=10 min) had no effect on cell volume, addingTMX at t=5 min partially inhibited the steady-state response, and addingTMX at the same time as applying the hypotonic stress both slowed therate of initial slowing and abolished the RVD. This confirms the conceptthat tamoxifen can inhibit swelling-activated chloride channels of theNPE cells, but selectively enhances the stimulatory effect of ATP onchloride channels by the PE cells.

[0034] FIGS. 12A-12B show the dependence on Cl⁻ of the synergisticshrinkage triggered by tamoxifen and ATP in PE cells. Gramicidin D (5μM) was present in all suspensions to provide an exit pathway for K⁺ Asshown in FIG. 12A, the simultaneous application of 6 μM tamoxifen and 10μM ATP produced isosmotic shrinkage in the presence of Cl⁻ to asteady-state value (v_(∞)) of 90.6%±1.1% with a time constant (τ) of13.2±3.2 min (N=4, P<0.01), but not in its absence. FIG. 12B, shows theeffect of Cl⁻-channel blockers on ATP, tamoxifen-activated shrinkage. Inthe absence of inhibitors, the shrinkage was fit with v_(∞)=94.2%±0.3%and τ=4.4±0.8 min. The Cl⁻ channel blockers DIDS (500 μM) reduced andNPPB (100 μM) each abolished the synergistic shrinkage (N=4, P<0.01).

[0035] FIGS. 13A-13C. show the potential roles of histamine andmuscarinic receptors in PE cells As shown in FIG. 13A, in the presenceof 100 μM ATP, either 10 μM carbachol or 10 μM tamoxifen, but not 10 μMhistamine, enhanced shrinkage (N=4). The value of the fits were:v_(∞)=94.9±1.0%, τ=45.2±14.0 min (100 μM ATP alone); v_(∞)=94.1±0.6%,τ=6.5±2.3 min (ATP+10 μM TMX); v_(∞)=97.4±0.5%, τ=21.0±8.0 min (ATP+10μM histamine); v_(∞)94.2±0.4%, τ=5.6±1.2 min (ATP+10 μM carbachol).

[0036] In FIG. 13B, carbachol (10 μM) produced nearly the same degree ofshrinkage in the presence or absence of 100 μM ATP (N=3). This effectwas entirely abolished by preincubating for 2 min with 10 μM atropineand retaining atropine in the test suspension. The fits generated thefollowing values: v_(∞)=87.5±1.8%, τ=45.2±14.0 min (ATP alone);v_(∞)=88.4±0.5%, τ=7.2±0.9 min (ATP+TMX); v_(∞)=87.4±0.5%, τ=6.3±0.8 min(ATP+TMX+atropine); v_(∞)=91.2±1.6%, τ=13.4±5.2 min (ATP+atropine).

[0037] In FIG. 13C, atropine (10 μM) had no significant effect on thevolumetric response to the combined application of 100 μM ATP and 10 μMtamoxifen (N=4). The values of the fits were: v_(∞)=93.9±1.7%,τ=34.8±13.8 min (ATP alone); v_(∞)=89.6±1.9%, τ=11.3±4.7 min (ATP+TMX);and v_(∞)=87.8±1.5%, τ=15.1±3.6 min (ATP+TMX+atropine). The cellsexposed to ATP and atropine did not display statistically significantshrinkage.

[0038] FIGS. 14A-14B show the potential role of calcium/calmodulin inATP/tamoxifen-mediated cell shrinkage of PE cells. FIG. 14A shows theinteractions of the calcium/calmodulin inhibitor trifluoperazine (10 μM)with ATP (100 μM) and tamoxifen (10 μM) on cell volume (N=6). Thepresence of trifluoperazine reduced the response to the combinedapplication of ATP and tamoxifen. The trajectories displayingsignificant shrinkage were fit with: v_(∞)=94.8±0.4%, τ=2.2±10.8 min(ATP+TMX); v_(∞)=96.2±0.3%, τ=6.8±1.4 min (ATP+trifluoperazine);v_(∞)=95.7±0.4%, τ=3.9±1.2 min (ATP+TMX+trifluoperazine). Thetrifluoperazine enhanced the shrinkage produced by ATP alone (P<0.01),but also significantly reduced the response produced by ATP+TMX(P<0.05).

[0039]FIG. 14B shows the effects of trifluoperazine (10 μM) and ATP (100μM) on PE cell volume in the absence of tamoxifen (N=5). The shrinkagetriggered by trifluoperazine was the same, whether or not ATP waspresent (P>0.05). The values of the fits were: v_(∞)=99.5±0.002%,τ=0.5±0.06 min (Control); v_(∞)=96.8±0.5%, τ=8.3±3.7 min (ATP alone);v_(∞)=96.8±0.2%, τ=0.9±0.6 min (trifluoperazine alone); v_(∞)=96.7±0.3%,τ=0.5±1.5 min (ATP+trifluoperazine).

[0040]FIG. 15 shows the potential role of protein kinase C on ATP andtamoxifen-mediated PE cell shrinkage. The effect of 250 μM DiC₈ and 0.3μM staurosporine with 10 μM tamoxifen and 100 μM ATP on cell volume(N=4) was determined. Only the aliquots exposed to tamoxifen and ATPdisplayed shrinkage (v_(∞)=89.7±6.1%; τ=22.9±19.0 min). The analyseswere conducted only with the first 6 time points because of theunusually large shrinkage triggered by ATP+tamoxifen at 30 min.

[0041] FIGS. 16A-16B show the potential role of estrogen receptors ontamoxifen and ATP-mediated PE cell shrinkage. FIG. 16A shows theinteractions of 17β-estradiol (100 nM) with ATP (100 μM)and tamoxifen(10 μM) on cell volume (N=4). Estradiol together with ATP initiated asmall and slow shrinkage, different from the baseline null response toATP, itself (P<0.05). The maximal response obtained with tamoxifen andATP (v_(∞)=93.9±1.0%, τ=15.6±5.0 min) was significantly inhibited(P<0.05) by adding estradiol 2 min before initiating the measurements.

[0042]FIG. 16B shows the interactions of estradiol and tamoxifen on PEcell volume. At the same 100-nM concentration, the active(17β-estradiol) and inactive (17α-estradiol) forms of the estrogenexerted very small and opposite effects on the time course of cellshrinkage.

[0043] FIGS. 17A-17B show the effects of tamoxifen and ATP onintracellular Ca²⁺ of PE cells. In FIG. 17A, 100 μM ATP or 100 μMATP+tamoxifen was applied for 20 sec as indicated by the short blackbars. The drugs were washed off for 5 min between applications. Althoughthe response attenuates, the presence of tamoxifen does not alter themagnitude of the response. Tamoxifen alone had no significant effect onCa²⁺ _(i) (not shown)

[0044] In FIG. 17B, mean results from 7 experiments in which 20-secapplications of 100 μM ATP were alternated with 20-sec applications of100 μM ATP+10 μM TMX. In order to adjust for the attenuation, two setsof experiments were performed. In the first set illustrated in A, theorder of drugs was 1) ATP+TMX, 2) ATP, 3) ATP+TMX and 4) ATP. In thesecond type of experiment the order was inverted. The magnitude of theCa²⁺ response when the first application was ATP alone (A1) was comparedwith experiments in which the first application was with ATP+TMX (T1).Trials in which the second application was with ATP (A2) were comparedwith trials in which the second application was ATP+TMX (T2), andlikewise for the third and fourth applications. There was no significantdifference between any of the four pairs (p<0.1, n=3-4)

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] The ciliary epithelium of the eye is a bilayer, comprising adeeper layer of pigmented epithelial (PE) cells and a superficial layerof nonpigmented epithelial (NPE) cells (FIG. 1). The proposed mechanismof action of ATP, tamoxifen (TMX) and A3 receptor agonists, such asadenosine (Ado), in influencing aqueous humor secretion is shown inFIG. 1. ATP is released from PE and/or NPE cells. ATP is then convertedto adenosine by ecto-enzymes (ecto). The adenosine then binds to A₃receptors on NPE cells, resulting in opening of Cl⁻ channels. Thisresults in an increase in aqueous humor production and increasedintraocular pressure. In addition, simultaneous stimulation by ATP andtamoxifen activates Cl⁻ efflux from PE cells, leading to a net decreasein aqueous humour formation. ATP acts on P₂ receptors of PE cells,promotes opening of Cl⁻ channels, and a decrease in aqueous humorproduction resulting in decreased intraocular pressure.

[0046] The present invention includes the observation that the A₃subtype adenosine receptor antagonists (referred to herein as A₃antagonists) inhibit shrinkage of NPE cells as determined bymeasurements of cell volume in isoosmotic solution. This inhibition ofcell shrinkage implies a net reduction of secretion of aqueous humorthrough the NPE cell membrane which would result in a reduction ofintraocular pressure (FIG. 1). These A₃ receptors are present on humanand rabbit NPE cells and underlie the activation of NPE chloride (Cl⁻)channels by adenosine. In addition, it was found that the antiestrogentamoxifen, the calcium/calmodulin inhibitor trifluoperazine and themuscarinic agonist carbachol all promoted cell shrinkage in PE cells.The shrinkage of PE cells implies a stimulation of a net reabsorption ofaqueous humor through the PE cell membrane towards the stroma, whichwould result in a net reduction in aqueous humor formation and areduction in intraocular pressure (FIG. 1). Thus, these compounds, orrelated compounds, can be used to lower intraocular pressure as atreatment for glaucoma and other ocular conditions in which it isdesirable to lower intraocular pressure.

[0047] Measurements of short-circuit current across intact rabbitciliary epithelium, of cell volume in suspended cultured human NPEcells, and of whole-cell currents from patch-clamped cultured human andfresh bovine NPE cells have indicated that adenosine-receptor occupancystimulates Cl⁻ secretion in mammalian NPE cells (Carre et al., supra.,1997). As evidenced by the data presented in the examples below, theseeffects are mediated by A₃ receptors. A₃ receptors are present in bothhuman HCE cells (a cell line of human NPE cells) and rabbit ciliarybody. The A₃-selective agonist IB-MECA increased the short circuitcurrent across rabbit iris-ciliary body in the presence of Ba²⁺, achange consistent with an increased efflux of Cl⁻ from NPE cells. In thepresence of gramicidin to isolate the Cl⁻ conductance, IB-MECA causedhuman HCE cells to shrink in a dose-dependent manner; the K_(d) of^(˜)55 nM is consistent with a maximal stimulation of A₃ receptors incardiac myocytes at 100 nM IB-MECA (Shahidullah et al., Curr. Eye Res.,16:1006-1016, 1997). The highly specific A₃ agonist Cl-IB-MECA alsoproduced shrinkage of HCE cells in the presence of gramicidin.Gramicidin readily partitions into plasma membranes to form acation-selective pore and is widely used for studying volume regulation(Hoffmann et al., Interaction of Cell Volume and Cell Function, Lang etal., eds., Springer, Heidelberg, Germany, p. 188-248, ACEP Series 14).Under these conditions, release of cell Cl⁻ becomes the rate-limitingfactor in both hypo-(Civan et al., Invest. Ophthalmol. Vis. Sci.,35:2876-2886, 1994) and isosmotic cell shrinkage (Carre et al., supra.,1997).

[0048] The A₃ antagonists MRS 1097 and MRS 1191 were able to prevent theshrinkage induced by IB-MECA at concentrations far below their K_(i) forA₁ and A_(2A) receptors. The A₁ agonist CPA did not have a consistenteffect upon cell volume. The A_(2A) agonist CGS-21680 had no effect atlow concentrations. The effect of CGS-21680 on shrinkage was onlydetected at a concentration 500 fold higher than the K_(i) values forthe A₃ receptor, and this effect was blocked by the A₃ antagonistMRS-1191. The A₃ antagonists MRS 1097, MRS 1191 and MRS 1523 blocked theshrinkage produced by 10 μM adenosine; at the concentrations used, <20%of the A₁ and A_(2A) receptors could have been occupied by MRS 1097 and<1% of those receptors could have been blocked by MRS 1191 and MRS 1523.Together, these observations indicate that the adenosine-stimulatedactivation of Cl⁻ release by the HCE line of human NPE cells isprimarily mediated by occupancy of an A₃-subtype adenosine receptor.

[0049] Adenosine but not ATP shrinks nonpigmented ciliary epithelial(NPE) cells by activating Cl⁻ channels. Although adenosine had no effecton PE cells, PE cell volume was occasionally reduced by ATP, and wasalways reduced by simultaneous application of ATP with the antiestrogentamoxifen. Cultured bovine PE cells were studied volumetrically byelectronic cell sorting. ATP alone (≧3 μM) shrank ˜15% of thesuspensions, but had little/no effect in most suspensions. Whole cellpatch clamping indicated that this baseline response reflectedactivation of Cl⁻ permeant channels in a heterogeneous population ofcells. The antiestrogen tamoxifen (6-10 μM) enhanced the ATP-triggeredshrinkage, whether or not a baseline response to ATP was detected. Thiswas unexpected since swelling activated Cl⁻ channels are either blocked(in NPE cells) or unaffected (in PE cells) by tamoxifen. Tamoxifen initself exerted no consistent effect on PE-cell volume. The tamoxifen,ATP-activated shrinkage required Cl⁻ release since the response wasblocked by removing Cl⁻ and was inhibited by Cl⁻ channel blockers (NPPBand DIDS). The modulating effect of tamoxifen could have reflected ≧5actions of tamoxifen. Our data argue against actions of tamoxifen toinhibit PKC or calcium/calmodulin and on histamine or carbacholreceptors. The cooperative interaction between tamoxifen and ATP was notmediated by an enhanced rise in Ca²⁺. The results indicate thattamoxifen interacts synergistically with ATP to activate Cl⁻ release bythe PE cells. Tamoxifen may act in part by occupying plasma-membraneestrogen-binding sites.

[0050] The present results demonstrate that tamoxifen markedly enhancedthe effects of extracellular ATP on the transport properties of culturedbovine pigmented ciliary epithelial cells. The synergism wasparticularly clear in those preparations with little or no baselineresponse to ATP alone and was detected at ATP and tamoxifenconcentrations likely to be physiologically and clinically relevant. Inthe presence of tamoxifen, an approximately half-maximal response waselicited by 3 μM ATP, a concentration likely reached physiologically byATP release into the constrained space between the PE cells and theunderlying basement membrane (Mitchell et al., supra., 1998). Tamoxifenis used clinically as an antiestrogen by occupying nuclearestrogen-receptor sites (Klinge et al., Oncology Research, 4,:145-150,1992), and the concentrations applied here (6-10 μM) also appear to beclinically relevant (Stuart et al., Br. J. Cancer, 66, 833-839, 1992).

[0051] The precise signaling pathway involved in the modulating actionof tamoxifen is presently unknown. In addition to binding to nuclearestrogen receptors (Klinge et al., supra.), and blocking plasma-membraneswelling-activated Cl⁻ channels (Valverde et al., Pflügers Archiv. 425:552-554, 1993; Zhang et al., J. Clin. Investi. 94:1690-1697, 1994;Nilius et al., Pflügers Archiv. 428:364-371, 1994; Wu et al., J.Physiol., 491.3: 743-755, 1996), tamoxifen has been observed to affect:histamine receptors (Brandes et al. Biochem. Biophys. Res. Commun., 126:905-910, 1986), muscarinic receptors (Ben-Baruch et al. Mol. Pharmacol.,21: 287-293, 1981), activation by calcium/calmodulin (Lam, Biochem.Biophys. Res. Commun. 118: 27-32,1984), plasma-membrane estrogenreceptors (Hardy et al., FASEB J. 8: 760-765. 1994), and protein kinaseC activity (O'Brien et al., Cancer Res. 45:2462-2465, 1985). The currentdata indicate that four of these five latter effects play no role in thesynergism between tamoxifen and ATP: (1) because histamine does notalter the response of cell volume to ATP, histamine receptors appear tobe irrelevant in the present context. (2) Although carbachol itselfshrinks cell volume, the response is not synergistic with ATP, andatropine does not affect the tamoxifen/ATP synergistic effect. Thus,muscarinic receptors are not involved. (3) Protein kinase C (PKC)activity cannot be playing a major role since both activation andinhibition of enzymatic activity produced similar small reductions involume, 1-2 orders of magnitude smaller than that triggered by tamoxifenand ATP together. At the same concentrations used here, the PKCactivator DiC₈ and the PKC inhibitor staurosporine exert large andopposing actions on swelling-activated Cl⁻ channels of nonpigmentedciliary epithelial cells (Civan et al., Invest. Ophthalmol. Vis. Sci.35: 2876-2886, 1994). (4) Finally, calcium/camodulin antagonism isunlikely to mediate the synergistic effect since the shrinkage producedby another such antagonist (trifluoperazine) was independent of thepresence of ATP.

[0052] It should be emphasized that although tamoxifen is not actinglike carbachol or trifluoperazine, both of these compounds did reduce PEcell volume. Both carbachol and trifluoperazine reduced cell volume ontheir own, in the absence of ATP. Thus, compounds like trifluoperazine,which inhibit calcium/calmodulin, or substances like carbachol, whichstimulate muscarinic receptors, could be used to reduce the productionof aqueous humor.

[0053] The potential role of binding to plasma-membrane estrogenreceptors is less clear. Hardy et al. (supra.) have reported that thattamoxifen activates a large-conductance Cl⁻ channel with an EC₅₀˜15 μMapplied to NIH 3T3 fibroblasts, but only after stable transfection withMDR1 and after growth in colchicine for >24 hrs. In contrast, noconsistent effect of tamoxifen on cell volume was observed in theabsence of ATP. Both the active (17-β-estradiol) and inactive(17-α-estradiol) forms of the estrogen had only slight effects onvolume, but 17-β-estradiol did enhance the ATP-activated shrinkageslightly. Interestingly, the active (but not the inactive) form of theestrogen also reduced the synergistic effect elicited by tamoxifen andATP. This indicates that tamoxifen and estrogen compete for bindingsites and that tamoxifen is more effective than 17-β-estradiol inactivating these sites. Since the response to tamoxifen was relativelyrapid (within 2 min), it is likely that these receptors are located atthe plasma membrane.

[0054] ATP produced an elevation in the levels on intracellular Ca²⁺which attenuated with repeated application, but the response was notaffected by the inclusion of tamoxifen. This implies that: 1) the cellshrinkage produced synergistically by ATP and tamoxifen is not mediatedby a synergistic elevation in Ca²⁺, and 2) the presence of TMX does notaffect the attenuation of the Ca²⁺ response to ATP. This attenuation hasbeen reported previously in ciliary epithelial cells, and it has beensuggested that the attenuation is mediated by the inhibition of IP₃production by increasingly elevated levels of PKC (Shahidulla et al.,1997). The inability of tamoxifen to modify the rate of attenuationsupports the interpretation that tamoxifen does not act by modifying PKCin these cells.

[0055] Thus, cells derived from the pigmented ciliary epithelial celllayer can respond to extracellular ATP by releasing Cl⁻, and thisrelease is strongly modulated by tamoxifen. Using the fluorescent probequinacrine, intracellular stores of ATP have been identified in NPE andPE cells in the intact epithelium and in culture (Mitchell et al.,supra, 1998). The ATP can be released by both cell types (Mitchell etal., supra., 1998) and metabolized to adenosine by ectoenzymes (Mitchellet al., supra., 1998). Adenosine is known to activate Cl⁻ channels NPEcells (Carré et al., supra., 1997). Taken together with these previousobservations, the current information suggests that ATP can enhancefluid movement in both directions across the ciliary epithelium:increasing secretion by stimulating NPE cells (indirectly throughadenosine formation) to release Cl⁻ into the aqueous humor, andincreasing reabsorption by directly stimulating PE cells to release Cl⁻into the stroma of the ciliary processes. It is likely that one or moreadditional factors is necessary to coordinate these opposing purinergicactions to permit ATP to regulate net aqueous humor formation.

[0056] This putative coordination could be provided in at least twoways. In principle, ATP could be released heterogeneously throughout theepithelium. Little information is as yet available on this point, butATP release triggered by anisosmotic swelling appears to be comparablefor NPE and PE cells (Mitchell et al., supra., 1998). An alternativepossible mechanism would be for a regulator, like tamoxifen, to modifythe effects of purines on Cl⁻ release at the contralateral surfaces ofthe ciliary epithelium. In the absence of tamoxifen, physiologicconcentrations (˜3 μM) of adenosine stimulate Cl⁻ release by NPE cells(Carré et al., supra., 1997), whereas even higher concentrations of ATPusually had little effect on release by PE cells. Under theseconditions; ATP release would favor secretion. In contrast, in thepresence of 6-10 μM tamoxifen, at least some of the Cl⁻ channels of theNPE cells are blocked (Wu et al., supra.; FIG. 4), and ATP is far moreeffective in stimulating Cl⁻ release from PE cells. Under these latterconditions, ATP release is expected to favor reabsorption.

[0057] The data presented below demonstrates the ability of variousagents to block shrinkage of NPE cells and to promote shrinkage of PEcells. The net effect of these agents would be to reduce intraocularpressure in vivo. The use of four chemical classes of A₃ receptorantagonists for reduction of intraocular pressure is also contemplated:dihydropyridines, pyridines, pyridinium salts and triazoloquinazolines.These generic compounds are shown in Appendix A, along with the possiblesubstituents at each variable position of the compound. These classes ofcompounds are also described in PCT/W097/27177.

[0058] In addition to the particular A₃ receptor antagonists discussedin the examples below: MRS-1097 (3-ethyl5-benzyl-2-methyl-6-phenyl-4-styryl-1,4-(±)-dihydropyridine-3,5-dicarboxylate),MRS-1191 (3-ethyl5-benzyl-2-methyl-6-phenyl-4-phenylethynyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate)(Jiang et al., J. Med. Chem. 40:2596-2608, 1997), and MRS-1523 (Li etal., J. Med. Chem. 42:706-721, 1999), the use of any A₃ receptorantagonist or analog thereof to reduce intraocular pressure is withinthe scope of the invention. Other A₃ receptor antagonists for use in thepresent invention are described by Jacobson (Trends Pharmacol. Sci.19:184-191, 1998) and include MRS-1334 (3-ethyl 5-(4-nitrobenzyl)2-methyl-6-phenyl-4-phenylethynyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate)),MRS1067 (3,6-dichloro-2′-(isopropoxy)-4′-methylflavone), MRS-1220(9-chloro-2-(2-furyl)-5-phenylacetylamino[1,2,4]triazolo[1,5-c]quinazoline),L249313(6-carboxymethyl-5,9-dihydro-9-methyl-2-phenyl-[1,2,4]-triazolo[5,1-a][2,7]naphthyridine)and L268605 (3-(4-methoxyphenyl)-5-amino-7-oxo-thiazolo[3,2]pyrimidine),VUF8504 (4-methoxy-N-[2-(2-pyridinyl)quinazdin-4-yl]benzamide) and thelike.

[0059] In a particularly preferred embodiment, the A₃ antagonist2,4-diethyl-1-methyl-3-(ethylsulfanylcarbonyl)-5-ethyloxycarbonyl-6-phenylpyridiumiodide (MRS 1649, 11) is used to reduce intraocular pressure. Thesynthesis of this compound is described in Example 15. This3,5-diacyl-1,2,4-trialkyl-6-phenylpyridinium derivative displays auniquely high water solubility (43 mM) and can be extracted readily intoether. In addition, the prodrug form of this compound, the corresponding1-methyl-1,4-dihydropyridine, can be oxidized to form compound 11 invitro in the presence of a tissue homogenate. Thus, it is contemplatedthat prodrug forms of A₃ receptor antagonists (e.g., compounds 24 and25) can be administered to the eye which will then be converted to theactive antagonists which will reduce intraocular pressure.

[0060] The determination of whether a compound can act as an A₃ receptorantagonist can be determined using standard pharmacological bindingassays. Similarly, although the antiestrogen tamoxifen is exemplifiedherein, other antiestrogens are also contemplated, including, but notlimited to, 4-hydroxy tamoxifen, toremifine, icosifene, droloxifene,LY117018, ICI 164,384, ICI 182,780, RU 58,668, EM-139, EM-800. EM-652,GW 5638, and the like. Lowering of intraocular pressure with acombination of an antiestrogen and ATP, or any compound capable ofpromoting ATP release from NPE cells, is also contemplated. Finally,although the calcium/calmodulin antagonist trifluoperazine isexemplified herein, the use of any calmodulin antagonist for loweringintraocular pressure is also within the scope of the inventionincluding, but not limited to calmidazolium chloride, calmodulin bindingdomain, chlorpromazine HCl, melittin, phenoxybenzamine HCl,trifluoperazine dimaleate, W-5, W-7, W-12 and W-13. These compounds areavailable from Calbiochem, San Diego, Calif. The use of analogs of theabove-identified compounds for the reduction of intraocular pressure isalso within the scope of the present invention.

[0061] These agents can be used to treat ocular disorders resultingassociated with or caused by an increase in intraocular pressure, suchas glaucoma. The agents can be processed in accordance with conventionalmethods to produce medicinal agents for administration to mammals,preferably humans. The agents can be employed in admixture withconventional excipients, i.e. pharmaceutically acceptable organic orinorganic carrier substances suitable for parenteral, enteral (e.g.oral) or topical application which do not deleteriously react with theagents. Suitable pharmaceutically acceptable carriers include, but arenot limited to, water, salt solutions, alcohols, gum arabic, vegetableoils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates suchas lactose, amylose or starch, magnesium stearate, talc, silicic acid,viscous paraffin, perfume oil, fatty acid monoglycerides anddiglycerides, pentaerythritol fatty acid esters, hydroxymethylcellulose, polyvinyl pyrollidone, etc. The pharmaceuticalpreparations can be sterilized and, if desired, mixed with auxiliaryagents, e.g., lubricants, preservatives, stabilizers, wetting agents,emulsifiers, salts for influencing osmotic pressure, buffers, coloring,flavoring and/or aromatic substances and the like which do notdeleteriously react with the active compounds. They can also be combinedwhere desired with other active agents, e.g., vitamins.

[0062] For parenteral application, particularly suitable are injectable,sterile solutions, preferably oily or aqueous solutions, as well assuspensions emulsions, or implants, including suppositories. Ampules areconvenient unit dosages. For enteral application, particularly suitableare tablets, liquids, drops, suppositories or capsules. A syrup, elixiror the like can be used when a sweetened vehicle is employed. Sustainedor directed release compositions can be formulated, e.g. liposomes orthose wherein the active compound is protected with differentiallydegradable coatings, e.g. by microencapsulation, multiple coatings, etc.It is also possible to lyophilize the agents for use in the preparationof products for injection.

[0063] Topical administration is preferred. For topical application,there are employed nonsprayable forms, viscous to semi-solid or solidforms comprising a carrier compatible with topical application andhaving a dynamic viscosity preferably greater than water. Suitableformulations include, but are not limited to, solutions, suspensions,emulsions, creams, ointments, powders, liniments, salves, aerosols,etc., which are, if desired, sterilized or mixed with auxiliary agents,e.g., preservatives, stabilizers, wetting agents, buffers or salts forinfluencing osmotic pressure, ocular permeability, etc. For topicalapplication, also suitable are sprayable aerosol preparations whereinthe active ingredient, preferably in combination with a solid or liquidinert carrier material, is packaged in a squeeze bottle or in admixturewith a pressurized volatile, normally gaseous propellant, e.g. a freon.In a particularly preferred embodiment, the agent is formulated into apharmaceutical formulation appropriate for administration to the eye,including eye drops, gels and ointments.

[0064] For systemic administration, the dosage of the agents accordingto this invention generally is between about 0.1 μg/kg and 10 mg/kg,preferably between about 10 μg/kg and 1 mg/kg. For topicaladministration, dosages of between about 0.000001% and 10% of the activeingredient are contemplated., preferably between about 0.1% and 4%. Itwill be appreciated that the actual preferred amounts of agent will varyaccording to the specific agent being used, the severity of thedisorder, the particular compositions being formulated, the mode ofapplication and the species being treated. Dosages for a given host canbe determined using conventional considerations, e.g.,by customarycomparison of the differential activities of the subject compounds andof a known agent, e.g. by means of an appropriate, conventionalpharmacologic protocol. The agents are administered from less than onceper day (e.g., every other day) to four times per day.

[0065] Gramicidin, adenosine, 2-chloroadenosine, tamoxifen, ATP, 17α-and β-estradiol, DiC₈, carbachol, atropine, histamine, andtrifluoperazine were obtained from the Sigma Chemical Co. (St. Louis,Mo.). CPA (N⁶-cyclopentyl-adenosine), CGS-21680, IB-MECA, Cl-IB-MECA andMRS-1191 (3-ethyl 5-benzyl2-methyl-6-phenyl-4-phenylethynyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate)were obtained from Research Biochemicals International (Natick, Mass.).Fura-2 AM was bought from Molecular Probes (Eugene, Oreg.). MRS 1097,and MRS 1523 were provided by Drs. Kenneth A. Jacobson (NationalInstitutes of Health) and Bruce L. Liang (University of Pennsylvania).The compound Cl-IB-MECA (MH-C-7-08; Lot No. CMVIII-12) was provided byResearch Biochemicals International as part of the Chemical SynthesisProgram of the National Institute of Mental Health, Contract N01MH30003.DIDS [4,4′-diisothiocyano-2,2′-disulfonic acid] and fura-2 AM wereobtained from Molecular Probes, Inc. (Eugene, Oreg.). NPPB[5-nitro-2-(3-phenylpropylamino)benzoate] and staurosporine wereobtained from Biomol Research Laboratories, Inc. (Plymouth Meeting,Pa.).

[0066] Values are presented as the means±1 SE. The number of experimentsis indicated by the symbol N. The null hypothesis, that the experimentaland baseline measurements shared the same mean and distribution, wastested with Student's t-test and by the upper significance limits of theF-distribution, as indicated. The t-test was applied to compare thesignificance between single means or single fit parameters. TheF-distribution was applied to test whether the time course of volumemeasurements in different suspensions could reflect a single populationof data points.

EXAMPLE 1 Cell Culture

[0067] The HCE (human ciliary epithelial) cell line (Carre et al.,supra.) is an immortalized NPE cell line obtained from primary culturesof adult human epithelium. Cells were grown in Dulbecco's modifiedEagle's medium (DMEM, #11965-027, Gibco BRL, Grand Island, N.Y.) with10% fetal bovine serum (FBS, A-1115-L, HyClone Laboratories, Inc.,Logan, Utah) and 50 μg/ml gentamycin (#15750-011, Gibco BRL), at 37° C.in 5% CO₂ (Wax et al., Exp. Eye Res. 57:3057-3063, 1993). The growthmedium had an osmolality of 328 mOsm. Cells were passaged every 6-7 daysand were studied 8-13 days after passage, after reaching confluence.

[0068] For the tamoxifen experiments, The cells used were animmortalized PE-cell line from a primary culture of bovine pigmentedciliary epithelium. Cells were grown in Dulbecco's modified Eagle'smedium (DMEM, #11965-027, Gibco BRL, Grand Island, N.Y.; and 51-43150,JRH Biosciences, Lenexa, Kans.) with 10% fetal bovine serum (FBS,A-1151-L, HyClone Laboratories, Inc., Logan, Utah) and 50 μg/mlgentamycin (#15750-011, Gibco BRL), at 37° C. in 5% CO₂ (Yantorno etal., Exp. Eye Res. 49:423-437, 1989). The medium had an osmolality of328 mOsm. Cells were passaged every 6-7 days and, after reachingconfluence, were suspended in solution for study within 6-10 days afterpassage.

EXAMPLE 2 Measurement of Cell Volume in Isosmotic Solution

[0069] The volume of NPE cells was measured as the movement of fluidthat underlies a change in NPE cell volume, this is thought to be thesame as the movement of fluid which underlines the secretion of aqueoushumor (FIG. 1).

[0070] A 0.5-ml aliquot of the HCE cell suspension in DMEM was added to20 ml of each test solution, which contained (in mM): 110.0 NaCl, 15.0HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], 2.5 CaCl₂,1.2 MgCl₂, 4.7 KCl, 1.2 KH₂PO₄, 30.0 NaHCO₃, and 10.0 glucose, at a pHof 7.4 and osmolality of 298-305 mOsm. Parallel aliquots of cells werestudied on the same day. One aliquot usually served as a control, andthe others were exposed to different experimental conditions at the timeof suspension. The same amount of solvent vehicle (dimethylformamide,DMSO or ethanol) was always added to the control and experimentalaliquots. The sequence of studying the suspensions was varied topreclude systematic time-dependent artifacts (Civan et al., Exp. EyeRes. 54:181-191, 1992).

[0071] Cell volumes of isosmotic suspensions were measured with aCoulter Counter (model ZBI-Channelyzer II), using a 100 μm aperture(Civan et al., supra., 1994). As previously described (Wax et al.,supra., 1993), the cell volume (v_(C)) of the suspension was taken asthe peak of the distribution function. Cell shrinkage was fit as afunction of time (t) to a monoexpenential function:

v _(C) =v ₂₈+(v ₀ −v _(∞))−[e ^(−(t−t) _(⁰) ^()/τ)]  {1}

[0072] where v_(∞) is the steady-state cell volume, v₀ is the cellvolume at the first point (t₀) of the time course to be fit, and τ isthe time constant of the shrinkage. For purposes of data reduction, thedata were normalized to the first time point, taken to be 100% isotonicvolume. Fits were obtained by nonlinear least-squares regressionanalysis, permitting both v_(∞) and τ to be variables.

[0073] In previous studies demonstrating that adenosine causes isotoniccell shrinkage by activating Cl⁻ channels in NPE cells (Carre et al.,supra., 1997), the levels of adenosine used were sufficiently high toactivate A₁, A_(2A), A_(2B) or A₃ adenosine receptor subtypes (Fredholm,et al., Pharmacol. Rev. 46:143-156, 1994, Fredholm et al., TrendsPharmacol. Sci. 18:79-82, 1997; Klotz et al., Naunyn Schmiedebergs Arch.Pharmacol. 357:1-9, 1998). In order to differentiate among thesereceptors, the experiments were repeated in the present study using aseries of agonists and antagonists selective for these receptors. As wewished to identify the effects of these receptors specifically on Cl⁻channels, 5 μM gramicidin D was included in all solutions to eliminateany potential contribution from K⁺ channels. This ionophore readilypartitions into plasma membranes to form a cation-selective pore, and iswidely used for studying volume regulation (Hoffmann et al., inInteraction of Cell Volume and Cell Function, Lang et al., eds.,Springer, Heidelberg, Germany, pp. 188-248, (ACEP Series 14), 1993)).Under these conditions, release of cell Cl⁻ becomes the rate-limitingfactor in both hypo-(Civan et al., Exp. Eye Res. 54:181-191, 1992) andisoosmotic cell shrinkage (Carre et al., supra., 1997).

[0074] In the presence of gramicidin, the A₃ agonist IB-MECA caused thecells to shrink in a concentration-dependent manner (FIGS. 2A-2B).Least-squares analysis of the linearized Lineweaver-Burk plot generatedfrom monoexponential fits of these data indicates that the apparentK_(d) for the IB-MECA-induced shrinkage was 55±10 nM (FIG. 2C). IB-MECAis a highly selective agonist for the A₃ receptor; the K_(i) for the A₃receptor is 50 times lower than it is for A₁ or A_(2A) receptor(Gallo-Rodrigez et al, J. Med. Chem. 37:636-646, 1994; Jacobson et al.,supra., 1995; Jacobson et al., FEBS Lett. 336:57-60, 1993)). Cl-IB-MECAis even more specific for A₃ receptors, with a K_(i) for A₃ receptors2500 times lower than for A₁ receptors and 1400 times lower than forA_(2A) receptors. The ability of Cl-IB-MECA to induce cell shrinkage(FIG. 2D) further strengthens the hypothesis that stimulation of A₃receptors stimulates Cl⁻ channels.

[0075] It was also determined whether A₃-selective antagonists couldprevent the putative A₃-mediated shrinkage produced by IB-MECA. Parallelaliquots of suspensions were preincubated with MRS 1097, a selectiveA₃-selective antagonist with K_(i) values for the binding (in nM) tohuman A₁/A₂/A₃ receptors of 5,930/4,770/108.(Jacobson et al.,Neuropharmacol. 36:1157-1165, 1997). Preincubation for 2 min with 300 nMMRS 1097 blocked the isoomotic shrinkage characteristically triggered by100 nM IB-MECA (FIG. 3A). A second highly selective A₃ antagonist, MRS1191, (Jlang et al., J. Med. Chem. 39:4667-4675, 1996), with K_(i)values for the binding (in nM) to human A₁/A₂/A₃ receptors of40,100/>100,000/31.4 (Jacobson et al., supra.) was also used.Preincubation for 2 min with 100 nM MRS 1097 also prevented thesubsequent response to 100 nM IB-MECA (FIG. 3B). There was an indicationin the results of FIG. 3B that MRS 1191 might actually produce a smallamount of cell swelling. This was not a constant finding (FIG. 4B, andmay have reflected variations in the background level of A₃-receptoroccupancy.

[0076] The physiologic agonist reaching the adenosine receptors islikely to be the nucleoside adenosine itself, arising from release ofATP by the ciliary epithelial cells and ecto-enzyme activity (Mitchellet al., 1998, supra.). Adenosine triggers isoosmotic shrinkage ofcultured human NPE cells with an EC₅₀ of 3-10 μM (Civan et al., supra.,1997). In this concentration range, adenosine acts as a nonselectiveagonist of all four subtypes of the adenosine receptor (Fredholm et al.,supra., 1994; Fredholm et al., supra., 1997). As illustrated in FIG. 4,a 2 min preincubation with either 100 nM of the A₃-selective antagonistMRS 1191 (FIG. 4B) or 300 nM of the A₃-selective antagonist MRS 1097(FIG. 4A) blocked the shrinkage characteristically produced by 10 μMadenosine. MRS 1523, an A₃ antagonist with K_(i) values for the binding(in nM) to human A₁/A₂/A₃ receptors of 15,600/2,050/19 (Li et al., J.Med. Chem. 41:3186-3201, 1998) also eliminated the actions of adenosine.

[0077] The ability of specific A₃ antagonists to inhibit the response tothe nonspecific adenosine suggests that the contribution of the otherreceptors to Cl⁻ channel activation was minimal. To test this further,the effect of A₁ and A_(2A) agonists were tested. CPA is an A₁-selectiveagonist with a K_(i) for the A₁-receptor of 0.6 nM (31). However, CPAproduced no significant shrinkage at 30 nM and 1 μM (data not shown,N=3) and 3 μM (FIG. 5A). A small slow effect of uncertain significancewas detected at the intermediate concentration of 100 nM (FIG. 5A). Somecross-reactivity with A₃ receptors might be expected, given the K_(i) ofCPA for the A₃-subtype of 43 nM (Klotz et al., supra.). CGS-21680 is awidely used A_(2A) agonist with an IC₅₀ value of 22 nM for theA_(2A)-receptor (Hutchison et al., J. Pharmacol. Exp. Ther. 251:47-55,1989, Jarvis et al., J. Pharmacol. Exp. Ther. 253:888-893, 1989).CGS-21680 had no detectable effect at 100-nM concentration (FIG. 5B),but did trigger isoosmotic shrinkage at a 30-fold higher concentration(3 μM) (FIG. 5C). However, the K_(i) for the CGS-21680 at the A₃receptor is 67 nM (Klotz et al., supra.) and thus CGS-21680 could havebeen acting though either A_(2A) receptors or A₃ receptors at the higherconcentration. To distinguish between these possibilities, wepreincubated parallel aliquots of suspensions with the antagonist 100 nMMRS 1191. MRS 1191 prevented the shrinkage produced by the highconcentration of CGS-21680 (FIG. 5C, P<0.01, F-test), indicating thatthe shrinkage observed was mediated by cross-reactivity with A₃receptors. As there are presently no high-affinity A_(2B) agonists(Klotz et al., supra.), the contribution of A_(2B) receptor stimulationwas not pursued, although the ability of A₃ antagonists to inhibit theresponse to 10 μM adenosine (FIG. 5) argues against a role for theA_(2B) receptor. For example, MRS1191 at 10 μM did not displaceradioligand binding to recombinant human A_(2B) receptors, thus it is atruly selective A₃ antagonist.

EXAMPLE 3 Effects of IB-MECA on Free Intracellular Calcium Levels

[0078] In other cells, stimulation of the A₃-receptor can lead to anelevation of intracellular Ca²⁺ (Kohno et al., supra.), so intracellularCa²⁺ was monitored in HCE cells to provide an additional physiologicassay for the presence of A₃ receptors. HCE cells grown on coverslipsfor 24-48 hrs were loaded with 1-5 μM fura-2 AM for 30-45 min at roomtemperature. The cells were subject to a post-incubation interval of20-40 minutes at room temperature before recording began. The coverslipswere mounted on a Nikon Diaphot microscope and visualized with a ×40oil-immersion fluorescence objective. The emitted fluorescence (510 nm)from 10-12 confluent cells was acquired at a sampling frequency of 1 Hzfollowing excitation at 340 nm and 380 nm, and the ratio was determinedwith a Delta-Ram system and Felix software (Photon TechnologyInternational Inc., Princeton, N.J.).Cells were perfused with anisotonic solution consisting of (in mM) 105 NaCl, 6 HEPES (acid), 4HEPES (Na⁺), 2 CaCl₂, 1 MgCl₂, 4 KCl, 5 glucose and 90 mannitol, at anosmolality of 327 mOsm, pH 7.4. The ratio of light excited at 380 nm vs.340 nm was converted into Ca²⁺ concentration using the followingequation (Grynkiewicz et al., J. Biol Chem. 260:3440-3550, 1985):$\begin{matrix}{\left\lbrack {Ca}^{2 +} \right\rbrack = {K_{d}*\left( \frac{\left( {R - {R\quad \min}} \right)}{\left( {{R\quad \max} - R} \right)} \right)\left( \frac{S_{2}f}{S_{2}b} \right)}} & \left\{ 2 \right\}\end{matrix}$

[0079] where R_(min) and R_(max) are the ratio of fluorescence at 340 nMvs. 380 nM in the absence of Ca²⁺ and in the presence of saturatingCa²⁺, respectively. R is the ratio measured experimentally. The S₂f andS₂b are the fluorescence emitted at 380 nM in the Ca²⁺ free and Ca²⁺bound states respectively. An in situ K_(d) value for fura-2 of 350 nMwas used (32). R_(min) was obtained by bathing cells in a Ca²⁺ freeisotonic solution containing 10 mM EGTA and 10 μM ionomycin. R_(max) wasobtained by bathing the cells in isotonic solution with 10 mM Ca²⁺ and10 μM ionomycin. Both calibration solutions were maintained at pH 8.0 tofacilitate Ca²⁺ exchange through ionomycin. Background fluorescenceobtained from confluent HCE cells in the absence of Fura-2 wassubtracted from all traces. Mean values of R_(min) and R_(max) were usedto obtain the mean responses for a set of experiments. Data wereanalyzed using a one-sided unpaired t-test.

[0080] Superfusion of HCE cells with 100 nM IB-MECA produced asustained, repeatable and frequently reversible increase in theintracellular Ca²⁺ concentration (FIG. 6). The increase in Ca²⁺ wasdependent upon concentration, with 100 nM IB-MECA leading to a mean riseof 17±5 nM Ca²⁺ ((p<0.01, N=8) while 1 μM IB-MECA intracellular Ca²⁺ by22±6 nM (p<0.05, N=3). Although these changes were relatively small,they were sustained, suggesting that these increases in Ca²⁺ could beresponsible for physiologic effects occurring on a time scale of minutesto hours.

EXAMPLE 4 Reverse Transcriptase (RT)-PCR Assays

[0081] RT-PCR amplifications of RNA from the human and rabbit NPE cellswere conducted using primers for the human A₃-type adenosine receptor.RNA was isolated from the HCE human NPE cell line using Trizol Reagent(Gibco BRL). Template was synthesized in vitro from the total RNA usingan RNA-PCR kit (Gene AMP, Perkin Elmer, Emeryville, Calif.). Thereaction mixture contained MuLV reverse transcriptase, an antisenseprimer specific for the A₃ subtype of adenosine receptor, and 1-5 μg oftotal RNA. Primers for the human A₃ receptor (Accession No. X76981) wereselected according to the Primer Select program (DNASTAR Inc., Madison,Wis.). The forward (sense) primer (nucleotides 914-937) was:5′-GCGCCATCATCTTGACATCTTTT-3′ (SEQ ID NO: 1). The reverse (antisense)primer (nucleotides 1373-1355) was: 5′-CTTGGCCCAGGCATACAGG-3′ (SEQ IDNO: 2). The cDNA was amplified by annealing the set of oligonucleotideprimers (0.2 μM) in a final volume reaction of 100 μl in an OmnigeneThermal Cycler (#480, HYBAID, Franklin, Mass.). The PCR reaction wasconducted for 35 cycles, each cycle comprising 1 min at 95° C., 1 min at55° C., and 1 min at 72° C. The final extension was prolonged by 7 minat 72° C. The PCR product was reamplified using the touchdown PCR methodwith fresh primers and TAQ polymerase, using an annealing temperatureranging from 58° C. to 48° C. The resulting PCR product wassize-fractionated by electrophoresis on 1% agarose gel. To sequence thePCR product, a band of the expected size (462 bp) was extracted from lowmelting-point agarose gel using a Qiaex II Agarose Gel Extraction kit(Qiaex, Calif.). The purified reaction product was directly sequenced onan ABI100 sequencer by the DNA Sequencing Facility at the Cell Center ofthe University of Pennsylvania and compared with the predicted sequenceusing a DNASTAR program.

[0082] The RT-PCR assay of rabbit A3 message was conducted in the sameway with the following changes. RNA was obtained from the tips of NewZealand White rabbit ciliary processes using Trizol Reagent, and wasreverse transcribed using 3-6 μg total RNA, MuLV reverse transcriptaseand oligo-dT primers. The reaction was carried out at 42° C. for 30minutes, followed by 5 minutes at 95° C. The PCR reaction andreamplification steps were performed using Amplitaq Gold (Perkin-Elmer,Foster City, Calif.) and 10% glycerol was included in thereamplification step. Specific primers for the rabbit A3 receptor wereselected from the rabbit A3 sequence (Accession No. U90718); the forwardprimer (nucleotides 147-167) was 5′-CAACCCCAGCCTGAAGACCAC-3′ (SEQ ID NO:3) while the reverse primer (nucleotides 608-587) was5′-TGAGAAGCAGGGGGATGAGAAT-3′ (SEQ ID NO: 4). Both PCR amplification andreamplification were performed for 35 cycles, each cycle consisting of 1min at 95° C., 1 min at 58.5° C. and 1 min at 72° C. A final extensioncycle of 7 minutes at 72° C. minutes completed the reaction.

[0083] The product of the PCR reamplification of rabbit tissue wascloned into the PCR-TOPO vector using the TOPO TA cloning kit(Invitrogen Corporation, Carlsbad, Calif.) following the manufacturer'sdirections. After transformation, plasmids were isolated using theWizard Plus Miniprep DNA Purification System (Promega Corporation,Madison, Wis.). The cloned plasmid was cut with EcoR I restrictionnuclease, and a band of approximately the expected size (479 bp) wasidentified by running the cut product on an agarose gel. The plasmid wassequenced from the Sp6 promoter site 80 base pairs proximal to the PCRproduct. The sequence was compared to the expected rabbit A₃ sequenceusing a DNASTAR program.

[0084] From the RT-PCR amplifications of human NPE cells using primersfor the human A₃ receptor, a fragment of the expected 462-bp size wasobtained, and was enhanced by direct PCR amplification of the product.The sequence obtained from the reamplified product was compared to thesequences of known human adenosine receptors using the DNASTAR program.The results displayed a 97.4% similarity to the published base sequencefor the A₃ receptor, whereas the similarity indices for the other knownadenosine-receptor subtypes were all <40% [37.9% for A₁ (Accession No.68485), 35.0% for A_(2A) (Accession No. 68486), and 36.7% for A_(2B)(Accession No. 68487)]. No product was detected whenreverse-transcriptase was excluded from the initial reaction mixture.

[0085] RT-PCR amplification was also conducted with rabbit ciliaryprocesses, using primers for the rabbit A₃-type adenosine receptor. TheRT-PCR product was reamplified, cloned and sequenced. The sequencedisplayed a 97.4% similarity with the published base sequence for therabbit A₃ receptor There was only 27.9% homology between rabbit A₁(Accession No. L01700) and A₃ receptors. Sequences are not yet availablefor the remaining A_(2A)- and A_(2B)-subtypes of adenosine receptors inthe rabbit. Our rabbit product also displayed 75.1% similarity to thehuman A₃ receptor but only <30% similarity indices for the other humanadenosine-receptor subtypes (28.2% for A₁, 27.7% for A_(2A) and 29.5%for A_(2B)). No product was detected when reverse-transcriptase wasexcluded from the reaction mixture.

EXAMPLE 5 Transepithelial Measurements

[0086] Adult male Dutch belted rabbits weighing 1.8-2.4 kg (Ace Animals,Boyertown, Pa.) were anesthetized with pentobarbital and sacrificed(Carre et al., J. Membr. Biol. 146:293-305, 1995). After enucleation,the iris-ciliary body (I-CB) was isolated as previously described (Carreet al., 1995, supra.). The experiments were in accordance with theResolution on the Use of Animals in Research of the Association forResearch in Vision and Ophthalmology.

[0087] The pupil and central iris were occluded with a Lucite disc, andthe iris-ciliary body was mounted between the two halves of a Lucitechamber (1). The annulus of exposed tissue provided a projected surfacearea of 0.93 cm². Preparations were continuously bubbled with95%O₂-5%CO₂ for maintenance of pH 7.4 in a Ringer's solution comprising(in mM): 110.0 NaCl, 10.0 HEPES (acid), 5.0 HEPES (Na⁺), 30.0 NaHCO₃,2.5 CaCl₂, 1.2 MgCl₂, 5.9 KCl, and 10.0 glucose, at an osmolality of 305mOsm. BaCl₂ (5 mM) was added to the solution to block K⁺ currents. Thetransepithelial potential was fixed at 0 mV, corrected for solutionseries resistance, and the short-circuit current was monitored on achart recorder. Data were digitally acquired at 10 Hz via a DigiData1200A converter and AxoScope 1.1 software (Axon Instruments, FosterCity, Calif.). Automatic averaging was performed with a reduction factorof 100 to achieve a final sampling rate of 6/min.

[0088] Adenosine in high concentration (100 μM) has been found toincrease the short-circuit current across the rabbit ciliary body (Carreet al., supra., 1997). We therefore tested whether a high concentration(30 μM) of the A₃ agonist IB-MECA also affected short-circuit current.At this concentration, the vehicle (dimethylformamide) itself exertssignificant effects (FIG. 7, lowest trajectory). We corrected for thesolvent effect in the following way. Solvent alone was initiallyintroduced (to 0.1%), followed by the same volume of solvent (to 0.2%)containing agonist, and ending with addition of a third identical volumeof solvent alone (to a final concentration of 0.3%). The reduction inshort-circuit current following the first addition of solvent was alwaysgreater than the third. In each of four experiments, we averaged thetime courses of the first and third additions to estimate the effect ofraising the solvent concentration without agonist from 0.1% to 0.2%during the experimental period. FIG. 7 presents the mean trajectory forthe averaged solvent effect, the uncorrected mean time course followingexposure to IB-MECA, and the mean trajectory±1 SE for thesolvent-corrected response. The experiments were performed in thepresence of 5 mM Ba²⁺ to minimize the contribution of K⁺ currents.IB-MECA produced a significant increase in the short-circuit current; anincrease in short-circuit current in the presence of Ba²⁺ suggests thatthe effect is mediated by activating a Cl⁻ conductance on thebasolateral membrane of the NPE cells. The sustained nature of thestimulation is consistent with the time course of the cell shrinkage inresponse to A₃ stimulation.

EXAMPLE 6 Volumetric Measurements and Analysis

[0089] The volume of PE cells was measured as the movement of fluid thatunderlies a change in PE cell volume, this is thought to be the same asthe movement of fluid which underlies the reabsorption of aqueous humor(FIG. 1).

[0090] After harvesting a single T-75 flask by trypsinization (Yantornoet al., supra.), a 0.5-ml aliquot of the bovine cell suspension in DMEM(or in Cl⁻-free medium, where appropriate), described in Example 1 wasadded to 20 ml of each test solution. The standard test solutioncontained (in mM) 110.0 NaCl, 15.0 HEPES[4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], 2.5 CaCl₂, 1.2MgCl₂, 4.7 KCl, 1.2 KH₂PO₄, 30.0 NaHCO₃, and 10.0 glucose, at a pH of7.4 and osmolality of 298-305 mOsm. The Cl⁻ free solution comprised:110.0 sodium methanesulfonate, 15.0 HEPES, 2.5 calcium methanesulfonate,1.2 MgSO₄, 4.7 potassium methanesulfonate, 1.2 KH₂PO₄, 30.0 NaHCO₃, and10.0 glucose, at a pH of 7.4 and osmolality of 294-304 mOsm. Parallelaliquots of cells were studied on the same day. One aliquot usuallyserved as a control, and the others were exposed to differentexperimental conditions at the time of suspension. The same amount ofsolvent vehicle (dimethylformamide, DMSO or ethanol) was always added tothe control and experimental aliquots. The sequence of studying thesuspensions was varied to preclude systematic time-dependent artifacts(Civan et al., 1994).

[0091] Cell volumes of isoosmotic suspensions were measured with aCoulter Counter (model ZBI-Channelyzer II), using a 100-μm aperture(Civan et al., 1992). As previously described (Yantorno et al., supra.),the cell volume (v_(C)) of the suspension was taken as the peak of thedistribution function. Cell shrinkage was fit as a function of time (t)to the simple exponential function:

v _(C)=(v ₀ −v _(∞))·(e ^(−t/τ))+v _(∞)  (1)

[0092] where v_(∞) is the steady-state cell volume, v₀ is cell volume att=0, and τ is the time constant of of the shrinkage. For purposes ofdata reduction; the data were normalized to the first time point, takento be 100% isotonic volume. The baseline isotonic value was 2488±203 fl(mean±SE, N=15). Fits were obtained by nonlinear least-squaresregression analysis, permitting both v_(∞) and τ to be variables (Carréet al., supra., 1997).

[0093] In approximately 15% of the volumetric studies, ATP producedshrinkage of the PE cells in suspension. As shown in FIG. 8A, theshrinkage was faster and larger after exposure to 100 μM than to 10 μMATP. In contrast to the results displayed in FIGS. 8A and 9A-C, ATPalone exerted very little effect on cell volume in ˜85% of the PEcell-suspensions studied over the concentration range 100 μM-10 mM(FIGS. 10A, 13A, 13C, 14A-C).

[0094] The clinically important, nonsteroidal antiestrogen tamoxifentriggered no consistent response in cell volume over a 30-min period ofobservation (FIGS. 10A-B). However, in the presence of tamoxifen, theresponse to ATP was strongly enhanced. The effect was most striking inthose preparations which displayed little or no shrinkage when exposedto 100 μM ATP (FIGS. 13A, 13C, 14A-C) or to 10 mM ATP (FIG. 10A).However, the volumetric response to ATP was also enhanced in cellpreparations responsive to ATP alone (FIG. 8B). No such interactiveresponse was observed between the corresponding nucleoside adenosine andtamoxifen (FIG. 10B).

[0095] At a constant tamoxifen concentration, ATP triggered detectableshrinkage at 10 μM, but not at 1 μM (FIG. 1C). At 3 μM ATP, theshrinkage was about half that noted with 10 μM ATP (both in the presenceof tamoxifen, FIG. 8B). The effects of ATP were comparable at 10 μM and1 mM (FIG. 8C). A concentration of 100 μM ATP was used in all of thesubsequent experiments to ensure that the concentration of ATP was notlimiting the rate of shrinkage. The choice of a tamoxifen concentrationof 6-10 μM was based on two considerations: 10 μM is the concentrationused in probing Cl⁻ channels in other cells (Wu et al., supra., 1996),and the concentration needed to produce a minimum detectable effect onthe response to swelling the current bovine PE cells is >2 μM and ≦6 μM(Mitchell et al., Invest. Ophthalmol. Vis. Sci.38 (Suppl.):S1042, 1997).

[0096] Several observations suggest that the ATP, tamoxifen-activatedshrinkage involved Cl⁻ release. First, the synergism occurred even whengramicidin was present to provide a constant pathway for K⁺ release(Civan et al., supra., 1994) (FIG. 11A), suggesting that the reductionin volume prompted by ATP and tamoxifen was due to the activation of ananionic conductance. Second, removal of Cl⁻ from the preincubation andtest solutions abolished the synergistic response (FIG. 11A). Third, theCl⁻-channel blockers NPPB (100 μM) (Wangemann et al., supra., 1986) andDIDS (500 μM) (Cabantchik et al., Am. J. Physiol., 262: C803-C827, 1992)both inhibited the volume reduction (FIG. 11B).

[0097] The synergistic stimulators effect of tamoxifen in shrinking PEcells was unexpected, given its inhibition of swelling-activated Cl⁻channels in many cells (Valverde et al., supra.; Zhang et al., supra.;Nilius et al., supra.), including NPE cells (Wu et al., supra, 1996),and the absence of any effect on swelling-activated Cl⁻ channels in PEcells (Mitchell et al., supra., 1997). Therefore, we reexamined theeffect of tamoxifen on the swelling-activated Cl⁻ channels of theimmortalized human NPE cells we have previously characterized (FIG. 12).After hypotonic swelling, the cell volume spontaneously fell (theregulatory volume decrease, RVD), reflecting the release of KCl andsecondarily water (Civan, et al., supra., 1994). Addition of tamoxifen10 min later, after the conclusion of the RVD, did not affect cellvolume, but addition 5 min after hypotonic suspension reduced themagnitude of the RVD. Inclusion of tamoxifen at the time of the initialhypotonic suspension completely abolished the RVD, consistent with theearlier report (Wu et al., supra., 1996) that tamoxifen blocksswelling-activated Cl⁻ channels of NPE cells. Tamoxifen also markedlyslowed the rate of hypotonic swelling (FIG. 12), raising the possibilitythat the antiestrogen also blocks the aquaporin-1 (AQP1) water channelsof the NPE cells (Stamer et al., Invest. Ophthalmol. Vis. Sci. 35:3867-3872, 1994; Lee et al., Current Topics in Membranes 45:105-134,1998).

[0098] The block of RVD in NPE cells by tamoxifen suggested thattamoxifen would reduce efflux of aqueous humor from NPE cells inaddition to its action of stimulating reabsorption from PE cells. Thus,tamoxifen could reduce aqueous humor production by two separatemechanisms.

EXAMPLE 7 Ruptured-Patch Whole-Cell Recording

[0099] Harvested cultured PE cells were resuspended and permitted tosettle and attach to glass coverslips, which were then transferred to aperfusion chamber (Carré et al., supra., 1997). Solutions were designedto isolate any C⁻ current activated by ATP. Thus both internal andexternal solutions were devoid of K⁺, and the cation reversal potentialwas >95 mV. The perfusate contained (in mM): 105.0 NaCl, 6.0 HEPES acid,4.0 HEPES Na⁺, 1.3 CaCl₂, 0.5 MgCl₂ and 90.0 mannitol (pH 7.4, 316 mOsm)and the micropipette-filling solution contained (in mM): 40.0 NCl, 135.0NMDG-OH, 95.0 CH₃SO₄ acid, 2.0 MgATP, 0.05 GTP, 1.1 EGTA and 0.55 CaCl₂(pH 7.2, 275 mOsm). Supplementary experiments were also conducted withfreshly-dissected bovine PE cells prepared by the method of Jacob et al.(Am. J. Physiol., 261:C1055-C1062, 1991), as previously described (Carréet al., supra., 1997). Cells were patched while still round, usually oneday after dissociation. The perfusate contained (in mM): 105.0 NaCl, 6.0HEPES acid, 4.0 HEPES Na⁺, 1.3 CaCl₂, 0.5 MgCl₂, 8.0 sucrose and 70.0mannitol (pH 7.4, 305 mOsm) and the micropipette-filling solutioncontained (in mM): 105.0 NMDG-Cl, 10.0 HEPES acid, 70.0 mannitol, 2.0MgATP, 0.01 GTP, 1.1 EGTA and 0.55 CaCl₂ (pH 7.2, 286 mOsm). Cells wereperfused with Na₂ATP dissolved directly into isotonic solution (1.0) orserially diluted (100 μM and 10 μM), while NPPB was diluted 1000:1 froma 100 mM stock in DMSO.

[0100] Data were acquired at 1 kHz using Axopatch-1B electronics andassociated headstage (Axon Instruments, Foster City, Calif.) andfiltered at 500 Hz with a Bessel filter. The micropipettes weredouble-pulled from Corning No. 7052 glass, coated with Sylgard and firepolished. The membrane potential was held without series-resistancecompensation at 0 mV and stepped to voltages over the range from −100 to+100 mV in 20 mV increments for 200 msec periods. The mean currentmeasured between 150-200 msec was used to obtain the patch-clamp dataused here. The data presented in FIG. 2 was obtained at −60 mV [theapproximate membrane potential (Green et al., Invest. Ophthalmol. Vis.Sci., 26:371-381, 1985)] to facilitate comparison with the volumetricdata.

[0101] ATP increased the current (FIG. 9A). The activated currents werelikely carried by Cl⁻ as solutions were chosen to minimize cationiccurrents, and the Cl⁻-channel blocker NPPB (100 μM) (Wangemann et al.,Pflügers Archiv. 407 (Suppl. 2):S128-S141, 1986) reduced theATP-enhanced currents at −60 mV by 56±11% (FIG. 9B, N=3). The activationof Cl⁻ channels was not restricted to the PE cell line, for ATP alsostimulated currents in fresh bovine PE cells (FIG. 9C) cells. Althoughcurrent was activated in both fresh and cultured cells, both cell typescontained a heterogeneous population with cultured cells responding in10/17 trials and fresh cells activated in 3/9 trials. Thus, ATP alonecan activate Cl⁻ curents directly, but only in half the cells.

EXAMPLE 8 Measurements of Intracellular Ca²⁺

[0102] Bovine PE cells grown on coverslips for 2-4 days were loaded with5 μM fura-2 AM for 30-45 min at room temperature, and then rinsed andmaintained in fura-free solution before beginning data acquisition. Thecoverslips were mounted on a Nikon Diaphot microscope and visualizedwith a ×40 oil-immersion fluorescence objective. The emittedfluorescence (510 nm) from 10-12 confluent cells was sampled at 1 Hzfollowing excitation at 340 nm and 380 nm, and the ratio determined witha Delta-Ram system and Felix software (Photon Technology InternationalInc., Princeton, N.J.). The ratio of light excited at 340 nm to that at380 nm was converted into Ca²⁺ concentration using the method ofGrynkiewicz et al. (J. Biol. Chem. 260:3440-3450, 1985). An in situK_(d) value for fura-2 of 350 nM was used (Negulescu et al., Meth.Enzymol. 192:38-81, 1990). R_(min) was obtained by bathing cells in aCa²⁺ free isotonic solution of pH 8.0 containing 10 mM EGTA and 5 μMionomycin. R_(max) was obtained by bathing the cells in isotonicsolution with 1.3 mM Ca²⁺ and 5 μM ionomycin. Calibration was performedseparately for each experiment. Baseline levels from PE cells in theabsence of fura-2 were subtracted from records to control forautofluorescence. Experiments were performed predominantly at roomtemperature, but several trials were performed at 37° C. using atemperature control unit from Warner Instrument Corp., (Hamden, Conn.).Cells were perfused with an isotonic solution containing (in mM) 105NaCl, 6 HEPES (acid), 4 HEPES (Na⁺), 1.3 CaCl₂, 1 MgCl₂, 4 KCl, 5glucose and 90 mannitol, at an osmolarity of 317 mOsm, pH 7.4. Tamoxifenwas stored as a 10 mM stock in ethanol for 2 days. In comparing theeffects of ATP and of ATP+tamoxifen, 0.1% ethanol was also added to thesolutions containing ATP alone.

EXAMPLE 9 Interaction with Histamine and Muscarinic Receptors

[0103] In addition to its actions on nuclear estrogen receptors (Klingeet al., supra.) and swelling-activated Cl⁻ channels (Valverde et al.,supra.; Zhang et al., supra.; Nilius et al., supra.; Wu et al., supra.),tamoxifen has been reported to produce multiple other effects.Experiments were conducted in order to address the possibility that oneor more of the following known actions of tamoxifen may be involved inATP, tamoxifen-activated shrinkage: interaction with histamine andmuscarinic receptors, antagonism of calcium/calmodulin, inhibition ofprotein kinase C, and antagonism of plasma- or nuclear membrane estrogenreceptors.

[0104] The antiestrogens are known to interact with histamine (Brandesetal., supra.) and muscarinic receptors (Ben-Baruch et al., supra.) inother preparations. FIG. 13A indicates that 10 μM histamine did notenhance the volumetric response to 100 μM ATP (N=4). Thenonmetabolizable muscarinic agonist carbachol did trigger a promptshrinkage in the presence of 100 μM ATP (FIG. 13A, N=4). However,carbachol triggered approximately the same response whether or not ATPwas present, and 10 μM atropine abolished that response (FIG. 13B, N=3).In contrast, tamoxifen had little effect in the absence of ATP (FIGS.8C, 10A and 10C), and 10 μM atropine did not alter the response to thecombined presence of tamoxifen and ATP (FIG. 13C, N=4). FIG. 13indicates that the volumetric actions of tamoxifen cannot be mediated byeither histamine or muscarinic receptors.

[0105] However, the results do show that carbachol can act alone toreduce PE cell volume. This suggests that carbachol, or similar agents,can stimulate fluid reabsorption by the PE cells and thus reduce the netproduction of aqueous humor.

EXAMPLE 10 Antagonism of Calcium/Calmodulin

[0106] Tamoxifen can inhibit calcium/calmodulin at the sameconcentration (10 μM) typically used to block Cl⁻ channels in NPE cells(Lam, supra.; Wu et al., supra., 1996). In PE cells, trifluoperazinetriggered a partial shrinkage of the bovine PE cells in the presence ofATP but this effect was not synergistic as similar effects were observedin the absence of ATP (FIG. 14). This suggests that althoughcalcium/calmodulin can modulate cell volume, it does not mediate thesynergistic action of tamoxifen. Thus, inhibitors of calcium/calmodulincan provide a separate additional route by which fluid efflux from thePE cells can be stimulated and the net production of aqueous humorreduced.

EXAMPLE 11 Protein Kinase C Inhibition

[0107] Tamoxifen can also inhibit protein kinase C (PKC), with a K_(i)of 5-100 μM depending on the assay system (O'Brien et al., supra.).However, inhibiting PKC activity with the PKC inhibitor staurosporineproduced a small, insignificant shrinkage in the presence of 100 μM ATP,substantially less that that produced by tamoxifen (FIG. 15). ActivatingPKC with DiC₈ in the presence of 100 μM ATP also had no significanteffect on cells volume. Thus, the synergistic effect of tamoxifen cannotbe mediated by its inhibition of baseline PKC activity.

EXAMPLE 12 Antagonism of Estrogen Receptors

[0108] In the presence of 100 nM 17β-estradiol, the response to thecombined application of ATP and tamoxifen was reduced, consistent withthe known antiestrogen action of tamoxifen (FIG. 16A, N=4, P<0.05,F-distribution). The 17β-estradiol also reduced the synergisticshrinkage produced by ATP and tamoxifen in another series of fourexperiments (P<0.01, F-distribution, data not shown), whereas theinactive estrogenic isomer 17α-estradiol had no significant effect(P>0.05, F-distribution). In the absence of tamoxifen, the 17α- and17β-estradiols exerted very small effects on cell volume (FIG. 16B,N=4). The data are consistent with the possibility that tamoxifen andestrogen compete for occupancy of the same population of receptors.

EXAMPLE 13 Potential Role of Ca²⁺

[0109] Non-pigmented ciliary epithelial cells show a synergisticelevation in free intracellular Ca²⁺ concentration (Ca²⁺ _(i)) uponsimultaneous presentation of certain drug pairs, and this synergism mayinvolve the activation of the G_(i) G-protein (Farahbakhsh et al., Exp.eye Res. 64:173-179, 1997). As ATP can activate G_(i) in a variety oftissues (Murthy et al., J. Biol. Chem. 273:4695-4704, 1998), it wasdetermined whether the signaling cascade for the synergistic shrinkageproduced by tamoxifen and ATP could reflect a synergistic change in Ca²⁺_(i). Tamoxifen (10 μM) itself triggered no significant change (Δ) inCa²⁺ _(i) (Δ=7±6 nM, N=4). Although Ca²⁺ _(i) increased in response toboth ATP and ATP+TMX, the comparison of the response was complicated bythe attenuation of the Ca²⁺ spike with repeated exposure, and thevariation between preparations. A 3-min application of either 100 μM ATPor 100 μM ATP+10 μM TMX usually produced an elevation in Ca²⁺but it wasdifficult to elicit a response of similar magnitude to a secondapplication 5 min later. Elevating the temperature to 37° C. did noteliminate the attenuation.

[0110] Nevertheless, it did prove possible to compare the magnitudes ofsuccessive Ca²⁺ responses when each drug application was limited toperiods of 20 sec (FIG. 17). Experiments were performed by alternating20-sec exposures to 100 μM ATP+10 μM TMX with 20 sec exposures to 100 μMATP alone (including 0.1% ethanol as a vehicle control for the TMX).Cells were washed in isotonic solution for 5 min between drugapplications, and 4-5 applications were possible per trial (FIG. 17A).The order of drug application shown in FIG. 10A, beginning first with100 μM ATP+10 μM TMX, is termed the T series. A parallel set ofexperiments termed the A series was performed which began with theapplication of ATP alone followed 5 min later by ATP+TMX.

[0111] To check for synergism while compensating for the attenuation,the responses to each application were compared for those experimentswhere ATP was first added (A series) with where ATP+TMX was first added(T series). Comparing the responses to successive applications of drugs,it is clear that there was no significant difference between the twoseries, whether ATP alone or ATP+TMX was added at a given point in time(FIG. 17B). The presence of TMX did not affect the size of the Ca²⁺response to ATP regardless of whether it was included in the first,second, third or fourth application (P>0.05 for applications 1-4,No=3.4). We conclude that ATP and tamoxifen did not produce asynergistic elevation in the level of intracellular Ca²⁺.

EXAMPLE 14 In vivo Animal Model

[0112] A rabbit model is used to determine the ability of A₃ subtypeadenosine receptor antagonists, antiestrogens and calmodulin antagoniststo reduce intraocular pressure. Ten normal New Zealand White rabbits areused for the study, sedated, and their intraocular pressure is measuredby standard optometric methods for several days at various times of theday to account for normal pressure variations. Once the average baselinepressure of each obtained, five rabbits are assigned to one of twogroups. In the first group, one eye is administered vehicle (e. g., cornoil or dimethyl sulfoxide (DMSO)) in the form of liquid drops and theother eye is left as an untreated control. In the second group, one eyeis administered vehicle plus test compound in the form of liquid drops,and the other eye is left as an untreated control. The test compound isadministered at a concentration ranging from about 1 nM to 100 mM to seea dose response relationship of intraocular pressure reduction.Intraocular pressure is then measured several hours after theadministration to determine reduction of intraocular pressure by thetest compound.

EXAMPLE 15 Synthesis of MRS-1649 and Related A₃ Receptor Antagonists

[0113] Materials. lodomethane was purchased from Fluka (Buchs,Switzerland). Iodoethane and 1-iodopropane were purchased from Aldrich(Milwaukee, Wis.). PBS (1× pH 7.4) was purchased from Biofluids, Inc.(Rockville, Md.). Starting 3,5-diacyl-2,4-dialkylpyridine anddihydropyridine derivatives were described previously (Li et al., J.Med. Chem. 41:3186-3201, 1998; Li et al., J. Med. Chem. 42:706-721,1999). All other materials were obtained from commercial sources.

[0114] Proton nuclear magnetic resonance spectroscopy was performed on aVarian GEMINI-300 spectrometer, and all spectra were obtained in CDCl₃.Chemical shifts (δ) relative to tetramethylsilane are given.Chemical-ionization (CI) mass spectrometry was performed with a Finnigan4600 mass spectrometer, and electron-impact (EI) mass spectrometry witha VG7070F mass spectrometer at 6 kV. Elemental analysis was performed byGalbraith Laboratories, Inc. (Knoxville, Tenn.) and/or AtlanticMicrolab, Inc. (Norcross, Ga.).

[0115] General Procedure for Preparation of Pyridinium Salt (10, 11,14-23) by Quaternary Amination of 3,5-Diacyl-2,4-DialkylpyridineDerivatives with Iodomethane: A mixture of a3,5-diacyl-2,4-dialkylpyridine derivative (14 mg, 0.038 mmol) andiodomethane (59 mg, 0.38 mmol) in 2 mL of anhydrous nitromethane wassealed in a Pyrex tube and was heated at 80° C. for 2 days. After themixture cooled to room temperature, the solvent and excess Mel wereremoved under reduced pressure to leave a yellow oil. It was applied toTLC separation [ethyl acetate:petroleum ether=1:4 (v/v) for the firstdevelopment; methanol:chloroform=1:5 (v/v) for a second development andethyl acetate:petroleum ether=1:1 (v/v) for a third development] and 9.5mg of the desired product (Pyridinium Salt, such as MRS 1649, 11) wasafforded as a yellow solid (yield: 49%). If methanol or acetone was usedas the solvent, the yield was much lower than with nitromethane.

[0116] HPLC results showed that 11 is free of the starting 2(2,4-diethyl-3-(ethylsulfanylcarbonyl)-5-ethyloxy-carbonyl-6phenylpyridine).The mobile phase used for the analysis consisted of methanol,acetonitrile and water (45:45:10). At a flow rate of 1.0 mL/min with a4.6×250 mm (internal diameter) reverse-phase 300 Å C-18 column operatedat ambient temperature, 11 had a retention time of 2.3 min (purity>99%).CHN analysis of 11: Calcd for C₂₂H₂₈INO₃S C: 51.47%, H: 5.50%, N: 2.73%.Found: C: 51.42%, H: 5.14%, N: 2.43%. HR-MS (FAB, m-b): Calcd forC₂₂H₂₈NO₃S (M⁺−I): 386.1790. Found: 386.1776. UV spectra was measuredusing a Beckman DU 640 Spectrophotometer. In methanol at ambienttemperature, 11 had a λ_(max)=203 nm, ε_(max)=6.65×10⁴ lmol⁻¹cm⁻¹;λ_(max)=224 nm, ε_(max)=3.30×10⁴ lmol⁻¹cm⁻¹; λ_(max)=288 nm,ε_(max)=9.62×10³ lmol⁻¹cm⁻¹.

[0117] The water solubility of 11 was measured by the following method.100 μL of de-ionized water was saturated with 5 mg 11 with heating.After cooling to room temperature and the disappearance of turbidity, 50μL of the clear supernatant was withdrawn and lyophilized to give 1.1 mgof 11. The water solubility of 11 was calculated to be 42.8 mM at roomtemperature.

1-Methyl-2-methyl-4-ethyl-3-(ethylsulfanylcarbonyl)-5-ethyloxycarbonyl-6-phenylpyridiniumIodide (10)

[0118]¹H-NMR δ: 0.88 (t, J=6.9 Hz, 3 H), 1.26 (t, J=7.5 Hz, 3 H), 1.33(t, J=7.5 Hz, 3 H), 2.69 (s, 3H), 2.79 (q, J=7.5 Hz, 2 H), 3.24 (q,J=7.5 Hz, 2 H), 4.01 (q, J=6.9 Hz, 2 H), 4.17 (s, 3 H), 7.49-7.53 (m, 3H), 7.65-7.68 (m, 2 H). MS (Cl/NH₃): m/z 500 (MH⁺), 358 (MH⁺−Me—I), 297(MH⁺−Me—I—SEt). K_(i)(hA₃)=379 nM.

1-methyl-2,4-Diethyl-3-(ethylsulfanylcarbonyl)-5-ethyloxycarbonyl-6-phenylpyridiniumIodide (MRS 1649, 11)

[0119]¹H-NMR δ: 0.86 (t, J=7.2 Hz, 3 H), 1.32 (t, J=7.8 Hz, 3 H), 1.41(t, J=7.8 Hz, 3 H), 1.44 (t, J=7.8 Hz, 3 H), 2.84 (q, J=7.8 Hz, 2 H),3.22 (q, J=7.8 Hz, 2 H), 3.44 (q, J=7.8 Hz, 2 H), 3.98 (q, J=7.2 Hz, 2H), 4.22 (s, 3 H), 7.56-7.62 (m, 3 H), 7.72-7.75 (m, 2 H). MS (Cl): m/z514 (MH⁺), 372 (MH⁺−Me—I). K_(i)(hA₃)=219 nM.

1-methyl-2,4-Diethyl-3-(ethylsulfanylcarbonyl)-5(2-fluoroethyloxycarbonyl)-6-phenylpyridinium Iodide (14)

[0120]¹H-NMR δ: 1.20 (t, J=7.5 Hz, 3 H), 1.39 (t, J=7.5 Hz, 3 H), 1.46(t, J=7.5 Hz, 3 H), 2.79 (q, J=7.5 Hz, 2 H), 2.99 (q, J=7.5 Hz, 2 H),3.27 (q, J=7.5 Hz, 2 H), 4.20 (s, 3 H), 4.28 (m, 2 H), 4.30-4.40 (m, 2H), 7.47-7.50 (m, 3 H), 7.64-7.67 (m, 2 H). MS (Cl/NH₃): m/z 390(MH⁺−Me—I). K_(i)(hA₃)=364 nM.

1-Methyl-2-ethyl-4-ethyl-3-(ethylsulfanylcarbonyl)-5-propyloxycarbonyl-6-phenylpyridiniumIodide (15)

[0121]¹H-NMR δ: 0.68 (t, J=7.8 Hz, 3 H), 1.25 (t, J=7.8 Hz, 3 H), 1.39(m, 2 H), 1.49 (t, J=7.8 Hz, 3 H), 1.57 (t, J=7.8 Hz, 3 H), 2.97 (q,J=7.8 Hz, 2 H), 3.28 (q, J=7.8 Hz, 2 H), 3.38 (q, J=7.8 Hz, 2 H), 4.07(t, J=6.9 Hz, 2 H), 4.21 (s, 3 H), 7.69-7.76 (m, 5 H). MS (Cl/NH₃): m/z386 (MH⁺−Me—I). K_(i)(hA₃)=483 nM.

1-Methyl-2-ethyl-4-propyl-3-(ethylsulfanylcarbonyl)-5-propyloxycarbonyl-6-phenylpyridiniumIodide (16)

[0122]¹H-NMR δ: 0.67 (t, J=7.8 Hz, 3 H), 1.04 (t, J=7.8 Hz, 3 H), 1.40(m, 2 H), 1.48 (t, J=7.8 Hz, 3 H), 1.55 (t, J=7.8 Hz, 3 H), 1.74 (m, 2H), 2.87 (t, J=7.8 Hz, 2 H), 3.27 (q, J=7.8 Hz, 2 H), 3.42 (q, J=7.8 Hz,2 H), 4.05 (t, J=7.8 Hz, 2 H), 4.20 (s, 3 H), 7.62-7.74 (m, 5 H). MS(Cl/NH₃): m/z 558 (M⁺+NH₄), 525 (M⁺−1-Me), 414 (M⁺−I), 369(M⁺−1-I—Me—Et). K_(i)(hA₃)=2.02 μM.

1-Methyl-2-ethyl-4-propyl-3-(3-fluoropropylsulfanylcarbonyl)-5-propyloxycarbonyl-6-phenylpyridiniumIodide (17)

[0123]¹H-NMR δ: 0.68 (t, J=7.8 Hz, 3 H), 1.04 (t, J=7.8 Hz, 3 H), 1.41(m, 2 H), 1.55 (t, J=7.8 Hz, 3 H), 1.73 (m, 2 H), 2.16 (m, 2 H), 2.88(m, 2 H), 3.35 (q, J=7.8 Hz, 2 H), 3.41 (t, J=6.9 Hz, 2 H), 4.06 (t,J=6.9 Hz, 2 H), 4.22 (s, 3 H), 4.55 (t, J=6.0 Hz, 1 H), 4.71 (t, J=6.0Hz, 1 H), 7.68-7.75 (m, 5 H).

[0124] MS (Cl/NH₃): m/z 432 (MH⁺−Me—I). K_(i)(hA₃)=465 nM.

1-Methyl-2-ethyl-4(2-acetylthioethyl)-3-(ethylsulfanylcarbonyl)-5-propyloxycarbonyl-6-phenylpyridiniumIodide (18)

[0125]¹H-NMR δ: 0.66 (t, J=7.5 Hz, 3 H), 1.37 (m, 2 H), 1.39 (t, J=7.8Hz, 3 H), 1.45 (t, J=7.2 Hz, 3 H), 2.35 (s, 3 H), 2.89-3.02 (m, 4 H),3.11 (m, 2 H), 3.28 (q, J=7.2 Hz, 2 H), 4.00 (t, J=6.9 Hz, 2 H), 4.23(s, 3 H), 7.53-7.55 (m, 3 H), 7.67-7.70 (m, 2 H). MS (Cl/NH₃): m/z 460(MH⁺−Me—I). K_(i)(hA₃)=538 nM.

1-Methyl-2-ethyl-4-(2-phthalimidoethyl)-3-(ethylsulfanylcarbonyl)-5-ethyloxycarbonyl-6-phenylpyridiniumIodide (19)

[0126]¹H-NMR δ: 0.94 (t, J=6.9 Hz, 3 H), 1.34 (t, J=7.2 Hz, 3 H), 1.48(t, J=7.2 Hz, 3 H), 2.94 (q, J=6.9 Hz, 2 H), 3.15 (t, J=7.8 Hz, 2 H),3.24 (q, J=7.2 Hz, 2 H), 4.01 (t, J=7.8 Hz, 2 H), 4.21 (s, 3 H), 4.25(q, J=7.2 Hz, 2 H), 7.51 (m, 3 H), 7.65 (m, 2 H), 7.78 (m, 2 H), 7.89(m, 2 ). MS (Cl/NH₃): m/z 517 (MH⁺−Me—I). K_(i)(hA₃)=1.25 μM.

1-Methyl-2-butyl-4-ethyl-3-(ethylsulfanylcarbonyl)-5-ethyloxycarbonyl-6-phenylpyridiniumIodide (20)

[0127]¹H-NMR δ: 0.90 (t, J=7.5 Hz, 3 H), 1.04 (t, J=7.5 Hz, 3 H), 1.29(t, J=7.5 Hz, 3 H), 1.34-1.43 (m, 2 H), 1.46 (t, J=7.5 Hz, 3 H), 1.84(m, 2 H), 2.76 (q, J=7.5 Hz, 2 H), 2.88 (t, J=7.5 Hz, 2 H), 3.16 (q,J=7.5 Hz, 2 H), 4.05 (q, J=7.5 Hz, 2 H) 4.26 (s, 3 H), 7.52-7.55 (m, 3H), 7.69-7.72 (m, 2 H). MS (Cl/NH₃): m/z 400 (MH⁺−Me—I). K_(i)(hA₃)=436nM.

1-Methyl-2-cyclobutyl-4-ethyl-3-(ethylsulfanylcarbonyl)-5-ethyloxycarbonyl-6-phenylpyridinium Iodide (21)

[0128]¹H-NMR δ: 0.99 (t, J=7.5 Hz, 3 H), 1.29 (t, J=7.5 Hz, 3 H), 1.45(t, J=7.5 Hz, 3 H), 1.88-1.97 (m, 1 H), 1.97-2.07 (m, 1 H), 2.18-2.32(m, 2 H), 2.53-2.65 (m, 2 H), 2.71 (q, J=7.5 Hz, 2 H), 3.13 (q, J=7.5Hz, 2 H), 3.81 (m, 1 H), 4.01 (q, J=7.5 Hz, 2 H), 4.23 (s, 3 H),7.54-7.56 (m, 3 H), 7.73-7.75 (m, 2 H). MS(Cl/NH₃): m/z 398 (MH⁺−Me—I).K_(i)(hA₃)=1.41 μM.

1-Methyl-2-(2-benzyloxyethyl)-4-propyl-3-(ethylsulfanylcarbonyl)-5-propyloxycarbonyl-6-phenylpyridiniumIodide (22)

[0129]¹H-NMR δ: 0.69 (t, J=7.2 Hz, 3 H), 1.02 (t, J=7.2 Hz, 3 H), 1.44(t, J=7.2 Hz, 3 H), 1.45 (m, 2 H), 1.68 (m, 2 H), 2.71 (m, 2 H), 3.17(q, J=7.2 Hz, 2 H), 3.24 (t, J=7.2 Hz, 2 H), 3.99 (t, J=7.2 Hz, 2 H),4.02 (q, J=7.2 Hz, 2 H), 4.25 (s, 3 H), 4.58 (s, 2 H), 7.29-7.35 (m, 5H), 7.53-7.56 (m, 3 H), 7.68-7.71 (m, 2 H). MS (Cl/NH₃): m/z 648 (MH⁺),506 (MH⁺−Me—I), 445 (MH⁺−Me—I—SEt). K_(i)(hA₃)=348 nM.

1-Methyl-2,4-diethyl-3(ethylsulfanylcarbonyl)-5-ethyloxycarbonyl)-6-cyclopentylpyridiniumIodide (23)

[0130]¹H-NMR δ: 1.10 (t, J=7.5 Hz, 3 H), 1.32 (t, J=7.5 Hz, 3 H), 1.41(t, J=7.5 Hz, 3 H), 1.44 (t, J=7.5 Hz, 3 H), 1.66 (m, 2 H), 1.95 (m, 7H), 2.62 (q, J=7.5 Hz, 2 H), 2.81 (q, J=7.5 Hz, 2 H), 3.97 (q, J=7.5 Hz,2 H), 4.28 (s, 3 H), 4.40 (q, J=7.5 Hz, 2 H). MS(Cl/NH₃): m/z 364(MH⁺−Me—I). K_(i)(hA₃)=695 nM.

1-Ethyl-2,4-diethyl-3-(ethylsulfanylcarbonyl)-5-ethyloxycarbonyl-6-phenylpyridiniumIodide (12)

[0131]¹H-NMR δ: 0.89 (t, J=7.5 Hz, 3 H), 1.28 (t, J=7.5 Hz, 3 H), 1.39(t, J=7.5 Hz, 3 H), 1.44 (t, J=7.2 Hz, 3 H), 2.79 (q, J=7.5 Hz, 2 H),2.94 (t, J=7.5 Hz, 3 H), 3.11 (q, J=7.5 Hz, 2 H), 3.34 (q, J=7.5 Hz, 2H). 4.02 (q, J=7.2 Hz, 2 H), 5.20 (q, J=7.5 Hz, 2 H), 7.52-7.58 (m, 3H), 7.68-7.71 (m, 2 H). MS (Cl/NH₃): m/z 372 (MH⁺−Et—I). K_(i)(hA₃)=577nM.

1-Propyl-2,4-diethyl-3-(ethylsulfanylcarbonyl)-5-ethyloxycarbonyl-6-phenylpyridiniumIodide (13)

[0132]¹H-NMR δ: 0.91 (m, J=7.2 Hz, 6 H), 1.29 (t, J=7.5 Hz, 3 H), 1.39(t, J=7.5 Hz, 3 H), 1.43 (t, J=7.2 Hz, 3 H), 2.44 (t, J=7.2 Hz, 3 H),2.79 (q, J=7.2 Hz, 2 H), 3.07 (q, J=7.5 Hz, 2 H), 3.38 (q, J=7.2 Hz, 2H), 4.01 (q, J=7.2 Hz, 2 H), 4.29 (q, J=7.2 Hz, 2 H), 5.22 (q, J=7.2 Hz,2 H), 7.51-7.54 (m, 3 H), 7.68-7.71 (m, 2 ). MS (Cl/NH₃): m/z 372(MH⁺−Pr—I). K_(i)(hA₃)=1.35 μM.

[0133] General Procedure for Preparation of 1-Methyl Dihydropyridines(24 and 25) by Alkylation of 3,5-Diacyl-2,4Dialkyl-1,4-DihydropyridineDerivatives with Iodomethane (Scheme 1): A solution of the appropriateDHP(2,4-diethyl-3(ethylsulfanylcarbonyl)-5-ethyloxycarbonyl-6-phenyl-1,4-dihydropyridine,15 mg 0.04 mmol) in 2 mL of anhydrous THF was treated with NaH (60%, 2mg, 0.08 mmol) at room temperature under stirring for 5 min. Theniodomethane (28 mg, 0.2 mmol) was added, and the reaction mixture wasstirred for another 10 min (monitor by TLC). At completion the reactionmixture was applied to TLC separation (ethyl acetate:petroleumether=1:19 v/v), and 11 mg of 24 was obtained (yield: 71%).

1-Methyl-2,4-diethyl-3-(ethylsulfanylcarbonyl)-5-ethyloxycarbonyl-6-phenyl-1,4-dihydropyridine(24)

[0134]¹H-NMR δ: 0.85 (t, J=7.2 Hz, 3 H), 0.93 (t, J=7.2 Hz, 3 H), 1.20(t, J=7.2 Hz, 3 H), 1.29 (t, J=7.2 Hz, 3 H), 2.74 (q, J=7.2 Hz, 2 H),2.85 (s, 3 H), 2.92 (q, J=7.2 Hz, 2 H), 3.07 (q, J=7.2 Hz, 2 H), 3.87(q, J=7.2 Hz, 2 H), 4.02 (t, J=7.2 Hz, 1 H), 7.20 (m, 2 H), 7.38-7.41(m, 3 H). MS (Cl/NH₃): m/z 388 (MH⁺).

1-Methyl-2,4-diethyl-3-(ethylsulfanylcarbonyl)-5-ethyloxycarbonyl-6-cyclopentyl-1,4-1,4-dihydropyridine(25)

[0135]¹H-NMR δ: 0.78 (t, J=7.8 Hz, 3 H), 1.11 (t, J=7.8 Hz, 3 H), 1.28(t, J=7.8 Hz, 3 H), 1.32 (t, J=7.8 Hz, 3 H), 1.46 (m, 2H), 1.60-1.78 (m,7H), 2.10 (m, 2 H), 2.60-2.67 (m, 1 H), 2.89 (q, J=7. Hz, 2 H),3.01-3.13 (m, 1 H), 3.15 (s, 3 H), 4.10 (t, J=6.0 Hz, 1 H), 4.13-4.26(m, 2 H). MS (Cl/NH₃): m/z 380 (MH⁺), 318 (M⁺−SEt, base).

[0136] Chemical Transformation of 24 to 11 through Oxidation with Iodine(Scheme 1): A solution of 24 (5 mg, 0.013 mmol) in 0.5 mL of drynitromethane was treated with iodine (10 mg, 0.040 mmol) at roomtemperature with stirring for 1 day (monitor by TLC). At completion thereaction mixture was applied to TLC separation (ethyl acetate:petroleumether=1:4 v/v for the first development then 1:1 for a seconddevelopment), and 2 mg of a yellow solid was obtained (yield: 30%), with¹H-NMR and MS data consistent with those of compound 11.

[0137] Oxidation of a 1-Methyl-1,4-Dihydropyridine Derivative (24) inthe Presence of Rat Brain Homogenate (Bodor et at., J. Med. Chem.26:528-534, 1983; Wu et al., J. Med. Chem. 32:1782-1788, 1999).

[0138] The rat brain homogenate was prepared by the following method.One rat (1 kg) was killed and the brain (weighing 2.26 g) was removed,and homogenized in 12 mL PBS (Biofluids, Inc., 1× pH 7.4). Thehomogenate was centrifuged at 12,000× rpm, and the supernatant was used.5 mg of 24 dissolved in 0.2 mL of DMSO was mixed with 10 mL of brainhomogenate (initial concentration of 1.27 mM), which was previouslyequilibrated to 37° C. in a water bath incubator, and shaking wascontinued at that temperature. Aliquots of 500 μL were withdrawn at 2,4, 8, 16, 32, 64, 128, 256, 768 min (2× each) from the test medium,added immediately to 3 mL of ice-cold ethyl ether, shaken vigorously,and placed in a freezer. When all samples had been collected, and theether layer of each sample was separated. After evaporation of theether, each residue was dissolved in 200 μL methanol, filtered throughWhatman 1 filter paper and analyzed by HPLC.

[0139] Pyridinium salt 11 could be generated through oxidation of thecorresponding reduced precursor,1-methyl-2,4-diethyl-3-(ethylsulfanylcarbonyl)-5-ethyloxycarbonyl-6-phenyl-1,4-dihydropyridine,24. The conversion of 24 to 11 by chemical means (iodine innitromethane) and during incubation at 37C with rat brain membranes, tosimulate in vivo conditions, was studied. The chemical conversionoccurred readily, and the time course of the biochemical oxidation wasrecorded. At regular time points, aliquots were removed from theincubation mixture, extracted with ether, and both the precursor and thepyridinium salt were assayed in the evaporated organic phase using HPLC.The oxidation occurred cleanly with a t_(1/2) of approximately 47 min.

[0140] The 1-methyl dihydropyridine 24 (precursor of MRS 1649) was foundto bind selectively to human A₃ adenosine receptors. At human A₃receptors, the K_(i) value was 379±122 nM (n=4). The K_(i) value at ratA₁ receptors was 28.4±2.5 μM (n=3). At rat A_(2A) receptors, the percentdisplacement of specific radioligand binding was 48±7% at 100 μM.Therefore to demonstrate the prodrug principle, i.e. an inactive prodrugthat could be converted to a selective adenosine antagonist, thecorresponding 6-cyclopentyl dihydropyridine 25 was prepared. The6-cyclopentyl analogue,1-methyl-2,4-diethyl-3-(ethylsulfanylcarbonyl)-5-ethyloxycarbonyl6-cyclopentyl-1,4-dihydropyridine (25), was shown to increase affinity at human A₃ receptors uponoxidation to the corresponding pyridinium salt (23). Compound 25 had aK_(i) value at human A₃ receptors of 6.40±0.78 μM, while 23 was an orderof magnitude more potent (K_(i) of 695 nM), suggesting a prodrug scheme.At rat A₁ receptors, both the precursor, compound 25 (34±1% displacementat 100 μM) and the oxidized product, 23 (K_(i) value 11.5 μM) bound onlyweakly.

[0141] Thus, in the present study, one reduced precursor, 25, was foundto be clearly less potent than the corresponding pyridinium salt, 23, inbinding to adenosine receptors, especially the A₃ subtype, and anotherprecursor, 24, was found to have the identical human A₃ receptoraffinity before and after oxidation. Thus, depending on the structure ofthe pyridine substitutents, one can determine the degree to which theantagonist requires preactivation of a prodrug form in order to acheiveantagonism of A₃ receptors.

EXAMPLE 16 Pharmacology

[0142] Adenylate cyclase assay. Adenylate cyclase assays were performedwith membranes prepared from Chinese hamster ovary (CHO) cells stablyexpressing either the human A₁ receptor or human A₃ receptor by themethod of Salomon et al.., (Anal. Biochem. 58:541-548, 1974) asdescribed previously with the following modifications (Jacobson et al.,Neuropharmacol., 36:1157-1165, 1997).4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724, 20 μM,Calbiochem, San Diego, Calif.) was employed to inhibitphosphodiesterases rather than papaverine, and the NaCl concentration inthe assay was 25 mM. Membranes were pretreated with 2 units/mL adenosinedeaminase, and the antagonist 11 (10 μM) at 30 C for 5 min prior toinitiation of the adenylate cyclase assay. Adenylate cyclase wasstimulated with forskolin (1 μM). Concentration-response data for theinhibition of adenylate cyclase activity by IB-MECA (human A₃ receptor)were obtained. Maximal inhibition of adenylate cyclase by IB-MECA at thehuman A₃ receptor correlated to ^(˜)60% of total stimulation,respectively. IC₅₀ values were calculated using InPlot (GraphPad, SanDiego, Calif.). K_(B) values were calculated as described. (Aluniakshanaet al., Brit. J. Pharmacol Chemother. 14:48, 1959).

[0143] Responses for agonist alone (0) or in combination with the A₃adenosine antagonist 11 (10 μM) were measured. IC₅₀ values were41.4±14.9 nM (IB-MECA alone), 1.08±0.19 μM (+11). Compound 11effectively antagonized the effects of an agonist in a functional A₃receptor assay, i.e. inhibition of adenylate cyclase in CHO cellsexpressing cloned human A₃ receptors.^(8,14) In this functional assay,IB-MECA inhibited adenylate cyclase via human A₃ receptors with an IC₅₀of 41.4±14.9 nM (n=3). In the presence of 10 μM of 11, the concentrationresponse curve was shifted 26-fold to the right, with an IC₅₀ of1.08±0.19 μM (n=3). From a Schild analysis (Alumakshana et al., supra.),a K_(B) value obtained for antagonism by 11 was 399 nM, i.e.approximately 1.8-times the K_(i) value obtained in binding to human A₃receptors.

[0144] Radioligand binding studies. Binding of[³H]R-N⁶-phenylisopropyladenosine ([³H]R-PIA) to A₁ receptors from ratcerebral cortex membranes and of[³H]-2-[4-[(2-carboxyethyl)phenyl]ethylamino]-5′-N-ethylcarbamoyladenosine([³H]CGS 21680) to A_(2A) receptors from rat striatal membranes wasperformed as described previously (Schwabe et al., Naunyn SchmiedebergArch. Pharmacol. 313:179-187, 1980; Jarvis et al., J. Pharmacol. Exp.Ther. 251:888-893, 1989) Adenosine deaminase (3 units/mL) was presentduring the preparation of the brain membranes, in a pre-incubation of 30min at 30° C., and during the incubation with the radioligands.Nonspecific binding was determined in the presence of 10 μM (A₁receptors) or 20 μM (A_(2A) receptors) 2-chloroadenosine.

[0145] Binding of[¹²⁵I]N⁶-(4-amino-3-iodobenzyl)-5′-N-methylcarbamoyladenosine([¹²⁵I]AB-MECA)²⁴ to membranes prepared from human embryonic kidney(HEK-293) cells stably expressing the human A₃ receptor (Salvatore etal., Proc. Natl Acad. Sci. U.S.A. 90:10365-10369, 1993), clone HS-21a(Receptor Biology, Inc., Beltsville, Md.) or to membranes prepared fromChinese hamster ovary (CHO) cells stably expressing the rat A₃ receptor(Zhou et al., Proc. Natl. Acad. Sci. U.S.A. 89:7432-7436, 1992) wasperformed as described at 4° C. (Olah et al., Mol. Pharmacol.i45:978-982, 1994). The assay medium consisted of a buffer containing 10mM Mg²⁺, 50 mM Tris, 3 units/mL adenosine deaminase, and 1 mM EDTA, atpH 8.0 (4 C). The glass incubation tubes contained 100 μL of themembrane suspension (0.3 mg protein/mL, stored at −80° C. in the samebuffer), 50 μL of [¹²⁵I]AB-MECA (final concentration 0.3 nM), and 50 μLof a solution of the proposed antagonist. Nonspecific binding wasdetermined in the presence of 100 μM N⁶-phenylisopropyladenosine (NECA).

[0146] All non-radioactive compounds were initially dissolved in DMSO,and diluted with buffer to the final concentration, where the amount ofDMSO never exceeded 1%. Incubations were terminated by rapid filtrationover Whatman GF/B filters, using a Brandell cell harvester (Brandell,Gaithersburg, Md.). The tubes were rinsed three times with 3 mL buffereach.

[0147] At least five different concentrations of competitor, spanning 3orders of magnitude adjusted appropriately for the IC₅₀ of eachcompound, were used. IC₅₀ values, calculated with the nonlinearregression method implemented in the InPlot program (Graph-PAD, SanDiego, Calif.), were converted to apparent K_(i) values using theCheng-Prusoff equation (Cheng et al., Biochem. Pharmacol. 22:3099-3108,1973) and K_(d) values of 1.0 nM ([³H]R-PIA); 15.5 nM ([³H]CGS 21680);0.59 nM and 1.46 nM ([¹²⁵I]AB-MECA at human and rat A₃ receptors,respectively).

1. A method for reducing intraocular pressure in an individual with anocular disorder, comprising the step of administering to said individualan effective intraocular pressure-reducing amount of a pharmaceuticalcomposition comprising an A₃ subtype adenosine receptor antagonist. 2.The method of claim 1, wherein said A₃ subtype receptor antagonist is adihydropyridine, pyridine, pyridinium salt or triazoloquinazoline. 3.The method of claim 1, wherein said A₃ subtype receptor antagonist isselected from the group consisting of MRS-1097, MRS-1191, MRS-1220,MRS-1523 and MRS-1649.
 4. The method of claim 1, wherein saidpharmaceutical composition is administered topically, systemically ororally.
 5. The method of claim 1, wherein said pharmaceuticalcomposition is an ointment, gel or eye drops.
 6. The method of claim 1,wherein said ocular disorder is glaucoma.
 7. A method for reducingintraocular pressure in an individual with an ocular disorder,comprising the step of administering to said individual an effectiveintraocular pressure-reducing amount of a pharmaceutical compositioncomprising an antiestrogen.
 8. The method of claim 7, wherein saidantiestrogen is tamoxifen.
 9. The method of claim 7, wherein saidpharmaceutical composition is administered topically, systemically ororally.
 10. The method of claim 7, wherein said pharmaceuticalcomposition is ointment, gel or eye drops.
 11. The method of claim 7,wherein said ocular disorder is glaucoma.
 12. A method for reducingintraocular pressure in an individual with an ocular disorder,comprising the step of administering to said individual an effectiveintraocular pressure-reducing amount of a pharmaceutical compositioncomprising a calmodulin antagonist.
 13. The method of claim 12, whereinsaid calmodulin antagonist is trifluoperazine.
 14. The method of claim12, wherein said pharmaceutical composition is administered topically,systemically or orally.
 15. The method of claim 12, wherein saidpharmaceutical composition is ointment, gel or eye drops.
 16. The methodof claim 12, wherein said ocular disorder is glaucoma.
 17. A method forreducing intraocular pressure in an individual with an ocular disorder,comprising the step of administering to said individual an effectiveintraocular pressure-reducing amount of a pharmaceutical compositioncomprising a prodrug which is converted into a A₃ subtype adenosinereceptor antagonist after said administering step.
 18. An A₃ subtypeadenosine receptor antagonist for use in reduction of intraocularpressure.
 19. The A₃ subtype adenosine receptor antagonist of claim 18,wherein said receptor antagonist is selected from the group consistingof MRS-1097, MRS-1191, MRS-1523 and MRS-1649.
 20. Use of an antiestrogenin the preparation of a medicament for the reduction of intraocularpressure.
 21. The use of claim 20, wherein said antiestrogen istamoxifen.
 22. A calmodulin antagonist for use in reduction ofintraocular pressure.
 23. The calmodulin antagonist of claim 22, whereinsaid calmodulin antagonist is trifluoperazine.
 24. A prodrug which isconverted into an A₃ receptor antagonist in vivo for use in reduction ofintraocular pressure.